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• W2134397312 abstract "To evaluate the role of the mitochondrial peripheral-type benzodiazepine receptor (PBR) in steroidogenesis, we developed a molecular approach based on the disruption of the PBR gene, by homologous recombination, in the constitutive steroid producing R2C rat Leydig tumor cell line. Inactivation of one allele of the PBR gene resulted in the suppression of PBR mRNA and ligand binding expression. Immunoblot and electron microscopic immunogold labeling analyses confirmed the absence of the 18-kDa PBR protein in the selected clone. Although mitochondria from the PBR-negative cells contained high levels of the constitutively expressed 30-kDa steroidogenic activity regulator protein, these cells produced minimal amounts of steroids compared with normal cells (5%). Moreover, mitochondria from PBR-negative cells failed to produce pregnenolone when supplied with exogenous cholesterol. Addition of the hydrosoluble cholesterol derivative, 22R-hydroxycholesterol, increased steroid production by the PBR-negative R2C cells, indicating that the cholesterol transport mechanism was impaired. Stable transfection of the PBR-negative R2C Leydig cells with a vector containing the PBR cDNA resulted in the recovery of the steroidogenic function of the cells. These data demonstrate that PBR is an indispensable element of the steroidogenic machinery, where it mediates the delivery of the substrate cholesterol to the inner mitochondrial side chain cleavage cytochrome P-450. To evaluate the role of the mitochondrial peripheral-type benzodiazepine receptor (PBR) in steroidogenesis, we developed a molecular approach based on the disruption of the PBR gene, by homologous recombination, in the constitutive steroid producing R2C rat Leydig tumor cell line. Inactivation of one allele of the PBR gene resulted in the suppression of PBR mRNA and ligand binding expression. Immunoblot and electron microscopic immunogold labeling analyses confirmed the absence of the 18-kDa PBR protein in the selected clone. Although mitochondria from the PBR-negative cells contained high levels of the constitutively expressed 30-kDa steroidogenic activity regulator protein, these cells produced minimal amounts of steroids compared with normal cells (5%). Moreover, mitochondria from PBR-negative cells failed to produce pregnenolone when supplied with exogenous cholesterol. Addition of the hydrosoluble cholesterol derivative, 22R-hydroxycholesterol, increased steroid production by the PBR-negative R2C cells, indicating that the cholesterol transport mechanism was impaired. Stable transfection of the PBR-negative R2C Leydig cells with a vector containing the PBR cDNA resulted in the recovery of the steroidogenic function of the cells. These data demonstrate that PBR is an indispensable element of the steroidogenic machinery, where it mediates the delivery of the substrate cholesterol to the inner mitochondrial side chain cleavage cytochrome P-450. Gonads, adrenal, placenta, and brain are the tissues in the body that have the ability to synthesize steroid hormones. Steroid synthesis begins with the conversion of the precursor cholesterol to pregnenolone by the cholesterol side chain cleavage cytochrome P-450 enzyme (P-450scc) 1The abbreviations used are: P-450scc, C27 cholesterol side chain cleavage cytochrome P-450; PBR, peripheral-type benzodiazepine receptor; StAR, steroidogenic activity regulator protein; PAGE, polyacrylamide gel electrophoresis; Ro5-4864, 4′-chlorodiazepam; PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; bp, base pair(s); kb, kilobase pair(s). 1The abbreviations used are: P-450scc, C27 cholesterol side chain cleavage cytochrome P-450; PBR, peripheral-type benzodiazepine receptor; StAR, steroidogenic activity regulator protein; PAGE, polyacrylamide gel electrophoresis; Ro5-4864, 4′-chlorodiazepam; PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; bp, base pair(s); kb, kilobase pair(s). and auxiliary electron transferring proteins, localized on inner mitochondrial membranes (1Hall P.F. Knobil E. Neil J. The Physiology of Reproduction. Raven Press, New York1988: 975-998Google Scholar, 2Simpson E.R. Waterman M.R. Can. J. Biochem. Cell Biol. 1983; 61: 692-707Crossref PubMed Scopus (148) Google Scholar, 3Kimura T. J. Steroid Biochem. 1986; 25: 711-716Crossref PubMed Scopus (44) Google Scholar, 4Jefcoate C.R. McNamara B.C. Artemenko I. Yamazaki T. J. Steroid Biochem. Mol. Biol. 1992; 43: 751-767Crossref PubMed Scopus (147) Google Scholar). The primary point of control in the acute stimulation of steroidogenesis, that occurs within minutes, by hormones is at the level of cholesterol transport, from intracellular sources to the inner mitochondrial membrane and the subsequent loading of cholesterol in the P-450scc active site (1Hall P.F. Knobil E. Neil J. The Physiology of Reproduction. Raven Press, New York1988: 975-998Google Scholar, 2Simpson E.R. Waterman M.R. Can. J. Biochem. Cell Biol. 1983; 61: 692-707Crossref PubMed Scopus (148) Google Scholar, 3Kimura T. J. Steroid Biochem. 1986; 25: 711-716Crossref PubMed Scopus (44) Google Scholar, 4Jefcoate C.R. McNamara B.C. Artemenko I. Yamazaki T. J. Steroid Biochem. Mol. Biol. 1992; 43: 751-767Crossref PubMed Scopus (147) Google Scholar). This hormone-dependent transport mechanism was shown to be mediated by cAMP and to be localized in the mitochondrion (1Hall P.F. Knobil E. Neil J. The Physiology of Reproduction. Raven Press, New York1988: 975-998Google Scholar, 2Simpson E.R. Waterman M.R. Can. J. Biochem. Cell Biol. 1983; 61: 692-707Crossref PubMed Scopus (148) Google Scholar, 3Kimura T. J. Steroid Biochem. 1986; 25: 711-716Crossref PubMed Scopus (44) Google Scholar, 4Jefcoate C.R. McNamara B.C. Artemenko I. Yamazaki T. J. Steroid Biochem. Mol. Biol. 1992; 43: 751-767Crossref PubMed Scopus (147) Google Scholar). Although a number of molecules have been proposed as potential candidates mediating this cholesterol transfer (3Kimura T. J. Steroid Biochem. 1986; 25: 711-716Crossref PubMed Scopus (44) Google Scholar, 4Jefcoate C.R. McNamara B.C. Artemenko I. Yamazaki T. J. Steroid Biochem. Mol. Biol. 1992; 43: 751-767Crossref PubMed Scopus (147) Google Scholar), no clear evidence has been presented on the identity of this mechanism. During the last decade, however, two cholesterol transport mechanisms have been identified and characterized as mediators of the acute stimulation of steroidogenesis by hormones, the PBR (5Papadopoulos V. Endocr. Rev. 1993; 14: 222-240Crossref PubMed Scopus (402) Google Scholar) and StAR (6Stocco D.M. Clark B.J. Endocr. Rev. 1996; 17: 221-244Crossref PubMed Scopus (926) Google Scholar) proteins.PBR is an 18-kDa protein discovered as a class of binding sites for benzodiazepines distinct from the GABAA neurotransmitter receptor (5Papadopoulos V. Endocr. Rev. 1993; 14: 222-240Crossref PubMed Scopus (402) Google Scholar). PBR are extremely abundant in steroidogenic cells (5Papadopoulos V. Endocr. Rev. 1993; 14: 222-240Crossref PubMed Scopus (402) Google Scholar) and found primarily on outer mitochondrial membranes (7Anholt R.R.H. Pedersen P.L. De Souza E.B. Snyder S.H. J. Biol. Chem. 1986; 261: 576-583Abstract Full Text PDF PubMed Google Scholar). PBR is a multimeric complex composed of the 18-kDa isoquinoline-binding protein and the 34-kDa pore-forming voltage-dependent anion channel protein, preferentially located on the outer/inner mitochondrial membrane contact sites (8McEnery M.W. Snowman A.M. Trifiletti R.R. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3170-3174Crossref PubMed Scopus (670) Google Scholar, 9Garnier M. Dimchev A. Boujrad N. Price M.J. Musto N.A. Papadopoulos V. Mol. Pharmacol. 1994; 45: 201-211PubMed Google Scholar, 10Papadopoulos V. Boujrad N. Ikonomovic M.D. Ferrara P. Vidic B. Mol. Cell. Endocr. 1994; 104: R5-R9Crossref PubMed Scopus (87) Google Scholar). Drug ligands of PBR, upon binding to the receptor, stimulate steroid synthesis in steroidogenic cells in vitro (11Papadopoulos V. Mukhin A.G. Costa E. Krueger K.E. J. Biol. Chem. 1990; 265: 3772-3779Abstract Full Text PDF PubMed Google Scholar, 12Ritta M.N. Calandra R.S. Neuroendocrinology. 1989; 49: 262-266Crossref PubMed Scopus (47) Google Scholar, 13Barnea E.R. Fares F. Gavish M. Mol. Cell. Endocr. 1989; 64: 155-159Crossref PubMed Scopus (57) Google Scholar, 14Amsterdam A. Suh B.S. Endocrinology. 1991; 128: 503-510Crossref Scopus (72) Google Scholar, 15Yanagibashi K. Ohno Y. Nakamichi N. Matsui T. Hayashida K. Takamura M. Yamada K. Tou S. Kawamura M. J. Biochem. (Tokyo). 1989; 106: 1026-1029Crossref PubMed Scopus (106) Google Scholar). Likewise, in vivo studies showed that high affinity PBR ligands increase steroid plasma levels in hypophysectomized rats (16Cavallaro S. Korneyev A. Guidotti A. Costa E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10598-10602Crossref PubMed Scopus (59) Google Scholar). Moreover, drug-induced decrease in adrenal PBR expression resulted in reduced circulating glucocorticoid levels in rats (17Amri H. Ogwuegbu S.O. Boujrad N. Drieu K. Papadopoulos V. Endocrinology. 1996; 137: 5707-5718Crossref PubMed Scopus (106) Google Scholar). Further in vitro studies on isolated mitochondria provided evidence that PBR ligands, drug ligands, or the endogenous PBR ligand, the polypeptide diazepam-binding inhibitor (5Papadopoulos V. Endocr. Rev. 1993; 14: 222-240Crossref PubMed Scopus (402) Google Scholar, 18Papadopoulos V. Amri H. Boujrad N. Cascio C. Culty M. Garnier M. Hardwick M. Li H. Vidic B. Brown A.S. Reversat J.L. Bernassau J.M. Drieu K. Steroids. 1997; 62: 21-28Crossref PubMed Scopus (335) Google Scholar), stimulate pregnenolone formation by increasing the rate of cholesterol transfer from the outer to the inner mitochondrial membrane (19Krueger K.E. Papadopoulos V. J. Biol. Chem. 1990; 265: 15015-15022Abstract Full Text PDF PubMed Google Scholar, 20Yanagibashi K. Ohno Y. Kawamura M. Hall P.F. Endocrinology. 1988; 123: 2075-2082Crossref PubMed Scopus (88) Google Scholar, 21Besman M.J. Yanagibashi K. Lee T.D. Kawamura M. Hall P.F. Shively J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4897-4901Crossref PubMed Scopus (231) Google Scholar, 22Papadopoulos V. Berkovich A. Krueger K.E. Costa E. Guidotti A. Endocrinology. 1991; 129: 1481-1488Crossref PubMed Scopus (191) Google Scholar). We also showed that hormone-stimulated steroidogenesis involves, at least in part, the participation of PBR (23Papadopoulos V. Nowzari F.B. Krueger K.E. J. Biol. Chem. 1991; 266: 3682-3687Abstract Full Text PDF PubMed Google Scholar). More recently, molecular modeling (24Bernassau J.M. Reversat J.L. Ferrara P. Caput D. Lefur G. J. Mol. Graph. 1993; 11: 236-245Crossref PubMed Scopus (96) Google Scholar, 25Papadopoulos V. Payne A.H. Hardy M.P. Russell L.D. The Leydig Cell. Cache River Press, Vienna, IL1996: 598-628Google Scholar) and in vitro reconstitution studies provided evidence that PBR may function as a mitochondrial cholesterol channel (18Papadopoulos V. Amri H. Boujrad N. Cascio C. Culty M. Garnier M. Hardwick M. Li H. Vidic B. Brown A.S. Reversat J.L. Bernassau J.M. Drieu K. Steroids. 1997; 62: 21-28Crossref PubMed Scopus (335) Google Scholar). 2H. Li and V. Papadopoulos, unpublished data. 2H. Li and V. Papadopoulos, unpublished data.StAR has been found only in gonadal and adrenal cells, where it is newly synthesized in response to trophic hormones and cAMP (26Pon L.A. Orme-Johnson N.R. J. Biol. Chem. 1986; 261: 6594-6599Abstract Full Text PDF PubMed Google Scholar, 27Stocco D.M. Kilgore M.W. Biochem. J. 1988; 249: 95-103Crossref PubMed Scopus (98) Google Scholar), as a cytoplasmic precursor protein of 37 kDa targeted to mitochondria (28Stocco D.M. Sodeman T.C. J. Biol. Chem. 1991; 266: 19731-19738Abstract Full Text PDF PubMed Google Scholar,29Epstein L.F. Orme-Johnson N.R. J. Biol. Chem. 1991; 266: 19739-19745Abstract Full Text PDF PubMed Google Scholar). The precursor further undergoes cleavage to produce the 30-kDa mitochondrial StAR protein and its phosphorylated counterpart (6Stocco D.M. Clark B.J. Endocr. Rev. 1996; 17: 221-244Crossref PubMed Scopus (926) Google Scholar). This protein processing is believed to occur at the level of the outer/inner mitochondrial membrane contact sites (6Stocco D.M. Clark B.J. Endocr. Rev. 1996; 17: 221-244Crossref PubMed Scopus (926) Google Scholar). StAR synthesis parallels the maximal capacity of the cells to produce steroids in response to trophic hormones, and expression of StAR in the absence of hormonal stimulation resulted in a 3-fold increase in progesterone production by MA-10 Leydig cells (30Clark B.J. Wells J. King S.R. Stocco D.M. J. Biol. Chem. 1994; 269: 28314-28322Abstract Full Text PDF PubMed Google Scholar, 31Clark B.J. Soo S.-C. Caron K.M. Ikeda Y. Parker K.L. Stocco D.M. Mol. Endocr. 1995; 9: 1346-1355Crossref PubMed Google Scholar). Both in vitro (30Clark B.J. Wells J. King S.R. Stocco D.M. J. Biol. Chem. 1994; 269: 28314-28322Abstract Full Text PDF PubMed Google Scholar, 31Clark B.J. Soo S.-C. Caron K.M. Ikeda Y. Parker K.L. Stocco D.M. Mol. Endocr. 1995; 9: 1346-1355Crossref PubMed Google Scholar) and in vivo (17Amri H. Ogwuegbu S.O. Boujrad N. Drieu K. Papadopoulos V. Endocrinology. 1996; 137: 5707-5718Crossref PubMed Scopus (106) Google Scholar) studies demonstrated that StAR is a hormone-regulated protein and mutations in StAR protein in humans is the cause of congenital lipoid adrenal hyperplasia, characterized by a deficiency in adrenal and gonadal steroid hormones (32Lin D. Sugawara T. Strauss J.F. Clark B.J. Stocco D.M. Saenger P. Rogol A. Miller W.L. Science. 1995; 267: 1828-1831Crossref PubMed Scopus (853) Google Scholar). Recent studies, however, demonstrated that StAR does not need to enter the mitochondria to stimulate steroidogenesis, suggesting that it functions by activating a mitochondrial receptor or transport mechanisms(s) (33Arakane F. Sugawara T. Nishino H. Liu Z. Holt J.A. Pain D. Stocco D.M. Miller W.L. Strauss III, J.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13731-13736Crossref PubMed Scopus (251) Google Scholar).R2C cells, derived from rat Leydig tumors (34Shin S. Yasumura Y. Sato G.H. Endocrinology. 1968; 82: 614-616Crossref PubMed Scopus (47) Google Scholar), maintain their in vitro capacity to synthesize steroids constitutively in a hormone-independent manner (34Shin S. Yasumura Y. Sato G.H. Endocrinology. 1968; 82: 614-616Crossref PubMed Scopus (47) Google Scholar, 35Freeman D.A. Endocrinology. 1987; 120: 124-132Crossref PubMed Scopus (48) Google Scholar). Thus, one can expect that constitutive steroidogenesis is driven by the unregulated expression of the hormonal mechanism controlling steroid synthesis or by an unknown separate mechanism. In 1994, we demonstrated that diazepam-binding inhibitor maintains constitutive steroidogenesis in R2C Leydig cells by binding to a mitochondrial higher affinity PBR which promotes continuous supply of cholesterol to the inner mitochondrial P-450scc (36Garnier M. Boujrad N. Ogwuegbu S.O. Hudson Jr., J.R. Papadopoulos V. J. Biol. Chem. 1994; 269: 22105-22112Abstract Full Text PDF PubMed Google Scholar). This higher affinity site was induced by the addition of constitutive cytosolic proteins on the mitochondrial PBR (36Garnier M. Boujrad N. Ogwuegbu S.O. Hudson Jr., J.R. Papadopoulos V. J. Biol. Chem. 1994; 269: 22105-22112Abstract Full Text PDF PubMed Google Scholar). In addition, Stocco and Chen (37Stocco D.M. Chen W. Endocrinology. 1991; 128: 1918-1926Crossref PubMed Scopus (102) Google Scholar) provided evidence that StAR was constitutively expressed in R2C cells. Thus, these cells provide the ideal system to examine the potential sequence of events mediating the mitochondrial cholesterol transport.In this report, we describe the use of gene targeting in the R2C steroidogenic cells to introduce a null mutation into one allele of the PBR gene. Characterization of the wild type and the mutant cell lines, as well as the mutant cells transfected with the PBR cDNA, reveals that PBR is indispensable for cholesterol transport into mitochondria and steroid synthesis.ConclusionWe have used the constitutive steroid-producing R2C Leydig cell to construct a PBR-null steroidogenic cell line using a method of gene inactivation based on targeted homologous recombination using selectable marker genes. In this PBR mutant cell line, steroidogenesis ceased due to the arrest of cholesterol transport across the outer mitochondrial membrane to the inner mitochondrial P-450scc. However, introduction of PBR in the PBR-negative R2C cells rescued steroidogenesis. These results are in agreement with our recent in vivo studies where a drug-induced decrease in adrenal PBR expression resulted in reduced circulating glucocorticoid levels in rats (17Amri H. Ogwuegbu S.O. Boujrad N. Drieu K. Papadopoulos V. Endocrinology. 1996; 137: 5707-5718Crossref PubMed Scopus (106) Google Scholar) and targeted mutagenesis of PBR showed its lethal effect on mouse embryo survival (18Papadopoulos V. Amri H. Boujrad N. Cascio C. Culty M. Garnier M. Hardwick M. Li H. Vidic B. Brown A.S. Reversat J.L. Bernassau J.M. Drieu K. Steroids. 1997; 62: 21-28Crossref PubMed Scopus (335) Google Scholar). Thus, these data provide unequivocal evidence on the determining role of PBR in cholesterol transport and steroid synthesis.Because PBR is a mitochondrial protein (5Papadopoulos V. Endocr. Rev. 1993; 14: 222-240Crossref PubMed Scopus (402) Google Scholar, 7Anholt R.R.H. Pedersen P.L. De Souza E.B. Snyder S.H. J. Biol. Chem. 1986; 261: 576-583Abstract Full Text PDF PubMed Google Scholar), PBR-null cells express high levels of StAR (present study), in vivo reduction of adrenal PBR expression and corticosterone synthesis occurred despite the presence of high levels of StAR (17Amri H. Ogwuegbu S.O. Boujrad N. Drieu K. Papadopoulos V. Endocrinology. 1996; 137: 5707-5718Crossref PubMed Scopus (106) Google Scholar), and StAR does not need to enter the mitochondria to stimulate pregnenolone formation (33Arakane F. Sugawara T. Nishino H. Liu Z. Holt J.A. Pain D. Stocco D.M. Miller W.L. Strauss III, J.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13731-13736Crossref PubMed Scopus (251) Google Scholar), we propose that PBR acts after StAR and that PBR may control or mediate the effect of StAR on mitochondrial steroid synthesis. In addition, the observations that (i) a cytosolic protein is required for the appearance of a higher affinity PBR coupled to constitutive (36Garnier M. Boujrad N. Ogwuegbu S.O. Hudson Jr., J.R. Papadopoulos V. J. Biol. Chem. 1994; 269: 22105-22112Abstract Full Text PDF PubMed Google Scholar) and hormone-induced (52Boujrad N. Gaillard J.-L. Garnier M. Papadopoulos V. Endocrinology. 1994; 135: 1576-1583Crossref PubMed Scopus (56) Google Scholar) steroid synthesis, (ii) PBR is coupled to voltage-dependent anion channel (9Garnier M. Dimchev A. Boujrad N. Price M.J. Musto N.A. Papadopoulos V. Mol. Pharmacol. 1994; 45: 201-211PubMed Google Scholar), a protein located on the outer/inner mitochondrial membrane contact sites, and (iii) the StAR precursor is targeted to mitochondria where it may be cleaved at the contact sites to enter the mitochondria (6Stocco D.M. Clark B.J. Endocr. Rev. 1996; 17: 221-244Crossref PubMed Scopus (926) Google Scholar), suggest that the coordinated interaction of PBR and StAR may be the key to the induction and maintenance of steroid synthesis. Gonads, adrenal, placenta, and brain are the tissues in the body that have the ability to synthesize steroid hormones. Steroid synthesis begins with the conversion of the precursor cholesterol to pregnenolone by the cholesterol side chain cleavage cytochrome P-450 enzyme (P-450scc) 1The abbreviations used are: P-450scc, C27 cholesterol side chain cleavage cytochrome P-450; PBR, peripheral-type benzodiazepine receptor; StAR, steroidogenic activity regulator protein; PAGE, polyacrylamide gel electrophoresis; Ro5-4864, 4′-chlorodiazepam; PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; bp, base pair(s); kb, kilobase pair(s). 1The abbreviations used are: P-450scc, C27 cholesterol side chain cleavage cytochrome P-450; PBR, peripheral-type benzodiazepine receptor; StAR, steroidogenic activity regulator protein; PAGE, polyacrylamide gel electrophoresis; Ro5-4864, 4′-chlorodiazepam; PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; bp, base pair(s); kb, kilobase pair(s). and auxiliary electron transferring proteins, localized on inner mitochondrial membranes (1Hall P.F. Knobil E. Neil J. The Physiology of Reproduction. Raven Press, New York1988: 975-998Google Scholar, 2Simpson E.R. Waterman M.R. Can. J. Biochem. Cell Biol. 1983; 61: 692-707Crossref PubMed Scopus (148) Google Scholar, 3Kimura T. J. Steroid Biochem. 1986; 25: 711-716Crossref PubMed Scopus (44) Google Scholar, 4Jefcoate C.R. McNamara B.C. Artemenko I. Yamazaki T. J. Steroid Biochem. Mol. Biol. 1992; 43: 751-767Crossref PubMed Scopus (147) Google Scholar). The primary point of control in the acute stimulation of steroidogenesis, that occurs within minutes, by hormones is at the level of cholesterol transport, from intracellular sources to the inner mitochondrial membrane and the subsequent loading of cholesterol in the P-450scc active site (1Hall P.F. Knobil E. Neil J. The Physiology of Reproduction. Raven Press, New York1988: 975-998Google Scholar, 2Simpson E.R. Waterman M.R. Can. J. Biochem. Cell Biol. 1983; 61: 692-707Crossref PubMed Scopus (148) Google Scholar, 3Kimura T. J. Steroid Biochem. 1986; 25: 711-716Crossref PubMed Scopus (44) Google Scholar, 4Jefcoate C.R. McNamara B.C. Artemenko I. Yamazaki T. J. Steroid Biochem. Mol. Biol. 1992; 43: 751-767Crossref PubMed Scopus (147) Google Scholar). This hormone-dependent transport mechanism was shown to be mediated by cAMP and to be localized in the mitochondrion (1Hall P.F. Knobil E. Neil J. The Physiology of Reproduction. Raven Press, New York1988: 975-998Google Scholar, 2Simpson E.R. Waterman M.R. Can. J. Biochem. Cell Biol. 1983; 61: 692-707Crossref PubMed Scopus (148) Google Scholar, 3Kimura T. J. Steroid Biochem. 1986; 25: 711-716Crossref PubMed Scopus (44) Google Scholar, 4Jefcoate C.R. McNamara B.C. Artemenko I. Yamazaki T. J. Steroid Biochem. Mol. Biol. 1992; 43: 751-767Crossref PubMed Scopus (147) Google Scholar). Although a number of molecules have been proposed as potential candidates mediating this cholesterol transfer (3Kimura T. J. Steroid Biochem. 1986; 25: 711-716Crossref PubMed Scopus (44) Google Scholar, 4Jefcoate C.R. McNamara B.C. Artemenko I. Yamazaki T. J. Steroid Biochem. Mol. Biol. 1992; 43: 751-767Crossref PubMed Scopus (147) Google Scholar), no clear evidence has been presented on the identity of this mechanism. During the last decade, however, two cholesterol transport mechanisms have been identified and characterized as mediators of the acute stimulation of steroidogenesis by hormones, the PBR (5Papadopoulos V. Endocr. Rev. 1993; 14: 222-240Crossref PubMed Scopus (402) Google Scholar) and StAR (6Stocco D.M. Clark B.J. Endocr. Rev. 1996; 17: 221-244Crossref PubMed Scopus (926) Google Scholar) proteins. PBR is an 18-kDa protein discovered as a class of binding sites for benzodiazepines distinct from the GABAA neurotransmitter receptor (5Papadopoulos V. Endocr. Rev. 1993; 14: 222-240Crossref PubMed Scopus (402) Google Scholar). PBR are extremely abundant in steroidogenic cells (5Papadopoulos V. Endocr. Rev. 1993; 14: 222-240Crossref PubMed Scopus (402) Google Scholar) and found primarily on outer mitochondrial membranes (7Anholt R.R.H. Pedersen P.L. De Souza E.B. Snyder S.H. J. Biol. Chem. 1986; 261: 576-583Abstract Full Text PDF PubMed Google Scholar). PBR is a multimeric complex composed of the 18-kDa isoquinoline-binding protein and the 34-kDa pore-forming voltage-dependent anion channel protein, preferentially located on the outer/inner mitochondrial membrane contact sites (8McEnery M.W. Snowman A.M. Trifiletti R.R. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3170-3174Crossref PubMed Scopus (670) Google Scholar, 9Garnier M. Dimchev A. Boujrad N. Price M.J. Musto N.A. Papadopoulos V. Mol. Pharmacol. 1994; 45: 201-211PubMed Google Scholar, 10Papadopoulos V. Boujrad N. Ikonomovic M.D. Ferrara P. Vidic B. Mol. Cell. Endocr. 1994; 104: R5-R9Crossref PubMed Scopus (87) Google Scholar). Drug ligands of PBR, upon binding to the receptor, stimulate steroid synthesis in steroidogenic cells in vitro (11Papadopoulos V. Mukhin A.G. Costa E. Krueger K.E. J. Biol. Chem. 1990; 265: 3772-3779Abstract Full Text PDF PubMed Google Scholar, 12Ritta M.N. Calandra R.S. Neuroendocrinology. 1989; 49: 262-266Crossref PubMed Scopus (47) Google Scholar, 13Barnea E.R. Fares F. Gavish M. Mol. Cell. Endocr. 1989; 64: 155-159Crossref PubMed Scopus (57) Google Scholar, 14Amsterdam A. Suh B.S. Endocrinology. 1991; 128: 503-510Crossref Scopus (72) Google Scholar, 15Yanagibashi K. Ohno Y. Nakamichi N. Matsui T. Hayashida K. Takamura M. Yamada K. Tou S. Kawamura M. J. Biochem. (Tokyo). 1989; 106: 1026-1029Crossref PubMed Scopus (106) Google Scholar). Likewise, in vivo studies showed that high affinity PBR ligands increase steroid plasma levels in hypophysectomized rats (16Cavallaro S. Korneyev A. Guidotti A. Costa E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10598-10602Crossref PubMed Scopus (59) Google Scholar). Moreover, drug-induced decrease in adrenal PBR expression resulted in reduced circulating glucocorticoid levels in rats (17Amri H. Ogwuegbu S.O. Boujrad N. Drieu K. Papadopoulos V. Endocrinology. 1996; 137: 5707-5718Crossref PubMed Scopus (106) Google Scholar). Further in vitro studies on isolated mitochondria provided evidence that PBR ligands, drug ligands, or the endogenous PBR ligand, the polypeptide diazepam-binding inhibitor (5Papadopoulos V. Endocr. Rev. 1993; 14: 222-240Crossref PubMed Scopus (402) Google Scholar, 18Papadopoulos V. Amri H. Boujrad N. Cascio C. Culty M. Garnier M. Hardwick M. Li H. Vidic B. Brown A.S. Reversat J.L. Bernassau J.M. Drieu K. Steroids. 1997; 62: 21-28Crossref PubMed Scopus (335) Google Scholar), stimulate pregnenolone formation by increasing the rate of cholesterol transfer from the outer to the inner mitochondrial membrane (19Krueger K.E. Papadopoulos V. J. Biol. Chem. 1990; 265: 15015-15022Abstract Full Text PDF PubMed Google Scholar, 20Yanagibashi K. Ohno Y. Kawamura M. Hall P.F. Endocrinology. 1988; 123: 2075-2082Crossref PubMed Scopus (88) Google Scholar, 21Besman M.J. Yanagibashi K. Lee T.D. Kawamura M. Hall P.F. Shively J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4897-4901Crossref PubMed Scopus (231) Google Scholar, 22Papadopoulos V. Berkovich A. Krueger K.E. Costa E. Guidotti A. Endocrinology. 1991; 129: 1481-1488Crossref PubMed Scopus (191) Google Scholar). We also showed that hormone-stimulated steroidogenesis involves, at least in part, the participation of PBR (23Papadopoulos V. Nowzari F.B. Krueger K.E. J. Biol. Chem. 1991; 266: 3682-3687Abstract Full Text PDF PubMed Google Scholar). More recently, molecular modeling (24Bernassau J.M. Reversat J.L. Ferrara P. Caput D. Lefur G. J. Mol. Graph. 1993; 11: 236-245Crossref PubMed Scopus (96) Google Scholar, 25Papadopoulos V. Payne A.H. Hardy M.P. Russell L.D. The Leydig Cell. Cache River Press, Vienna, IL1996: 598-628Google Scholar) and in vitro reconstitution studies provided evidence that PBR may function as a mitochondrial cholesterol channel (18Papadopoulos V. Amri H. Boujrad N. Cascio C. Culty M. Garnier M. Hardwick M. Li H. Vidic B. Brown A.S. Reversat J.L. Bernassau J.M. Drieu K. Steroids. 1997; 62: 21-28Crossref PubMed Scopus (335) Google Scholar). 2H. Li and V. Papadopoulos, unpublished data. 2H. Li and V. Papadopoulos, unpublished data. StAR has been found only in gonadal and adrenal cells, where it is newly synthesized in response to trophic hormones and cAMP (26Pon L.A. Orme-Johnson N.R. J. Biol. Chem. 1986; 261: 6594-6599Abstract Full Text PDF PubMed Google Scholar, 27Stocco D.M. Kilgore M.W. Biochem. J. 1988; 249: 95-103Crossref PubMed Scopus (98) Google Scholar), as a cytoplasmic precursor protein of 37 kDa targeted to mitochondria (28Stocco D.M. Sodeman T.C. J. Biol. Chem. 1991; 266: 19731-19738Abstract Full Text PDF PubMed Google Scholar,29Epstein L.F. Orme-Johnson N.R. J. Biol. Chem. 1991; 266: 19739-19745Abstract Full Text PDF PubMed Google Scholar). The precursor further undergoes cleavage to produce the 30-kDa mitochondrial StAR protein and its phosphorylated counterpart (6Stocco D.M. Clark B.J. Endocr. Rev. 1996; 17: 221-244Crossref PubMed Scopus (926) Google Scholar). This protein processing is believed to occur at the level of the outer/inner mitochondrial membrane contact sites (6Stocco D.M. Clark B.J. Endocr. Rev. 1996; 17: 221-244Crossref PubMed Scopus (926) Google Scholar). StAR synthesis parallels the maximal capacity of the cells to produce steroids in response to trophic hormones, and expression of StAR in the absence of hormonal stimulation resulted in a 3-fold increase in progesterone production by MA-10 Leydig cells (30Clark B.J. Wells J. King S.R. Stocco D.M. J. Biol. Chem. 1994; 269: 28314-28322Abstract Full Text PDF PubMed Google Scholar, 31Clark B.J. Soo S.-C. Caron K.M. Ikeda Y. Parker K.L. Stocco D.M. Mol. Endocr. 1995; 9: 1346-1355Crossref PubMed Google Scholar). Both in vitro (30Clark B.J. Wells J. King S.R. Stocco D.M. J. Biol. Chem. 1994; 269: 28314-28322Abstract Full Text PDF PubMed Google Scholar, 31Clark B.J. Soo S.-C. Caron K.M. Ikeda Y. Parker K.L. Stocco D.M. Mol. Endocr. 1995; 9: 1346-1355Crossref PubMed Google Scholar) and in vivo (17Amri H. Ogwuegbu S.O. Boujrad N. Drieu K. Papadopoulos V. Endocrinology. 1996; 137: 5707-5718Crossref PubMed Scopus (106) Google Scholar) studies demonstrated that StAR is a hormone-regulated protein and mutations in StAR protein in humans is the cause of congenital lipoid adrenal hyperplasia, characterized by a deficiency in adrenal and gonadal steroid hormones (32Lin D. Sugawara T. Strauss J.F. Clark B.J. Stocco D.M. Saenger P. Rogol A. Miller W.L. Science. 1995; 267: 1828-1831Crossref PubMed Scopus (853) Google Scholar). Recent studies, however, demonstrated that StAR does not need to enter the mitochondria to stimulate steroidogenesis, suggesting that it functions by activating a mitochondrial receptor or transport mechanisms(s) (33Arakane F. Sugawara T. Nishino H. Liu Z. Holt J.A. Pain D. Stocco D.M. Miller W.L. Strauss III, J.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13731-13736Crossref PubMed Scopus (251) Google Scholar). R2C cells, derived from rat Leydig tumors (34Shin S. Yasumura Y. Sato G.H. Endocrinology. 1968; 82: 614-616Crossref PubMed Scopus (47) Google Scholar), maintain their in vitro capacity to synthesize steroids constitutively in a hormone-independent manner (34Shin S. Yasumura Y. Sato G.H. Endocrinology. 1968; 82: 614-616Crossref PubMed Scopus (47) Google Scholar, 35Freeman D.A. Endocrinology. 1987; 120: 124-132Crossref PubMed Scopus (48) Google Scholar). Thus, one can expect that constitutive steroidogenesis is driven by the unregulated expression of the hormonal mechanism controlling steroid synthesis or by an unknown separate mechanism. In 1994, we demonstrated that diazepam-binding inhibitor maintains constitutive steroidogenesis in R2C Leydig cells by binding to a mitochondrial higher affinity PBR which promotes continuous supply of cholesterol to the inner mitochondrial P-450scc (36Garnier M. Boujrad N. Ogwuegbu S.O. Hudson Jr., J.R. Papadopoulos V. J. Biol. Chem. 1994; 269: 22105-22112Abstract Full Text PDF PubMed Google Scholar). This higher affinity site was induced by the addition of constitutive cytosolic proteins on the mitochondrial PBR (36Garnier M. Boujrad N. Ogwuegbu S.O. Hudson Jr., J.R. Papadopoulos V. J. Biol. Chem. 1994; 269: 22105-22112Abstract Full Text PDF PubMed Google Scholar). In addition, Stocco and Chen (37Stocco D.M. Chen W. Endocrinology. 1991; 128: 1918-1926Crossref PubMed Scopus (102) Google Scholar) provided evidence that StAR was constitutively expressed in R2C cells. Thus, these cells provide the ideal system to examine the potential sequence of events mediating the mitochondrial cholesterol transport. In this report, we describe the use of gene targeting in the R2C steroidogenic cells to introduce a null mutation into one allele of the PBR gene. Characterization of the wild type and the mutant cell lines, as well as the mutant cells transfected with the PBR cDNA, reveals that PBR is indispensable for cholesterol transport into mitochondria and steroid synthesis. ConclusionWe have used the constitutive steroid-producing R2C Leydig cell to construct a PBR-null steroidogenic cell line using a method of gene inactivation based on targeted homologous recombination using selectable marker genes. In this PBR mutant cell line, steroidogenesis ceased due to the arrest of cholesterol transport across the outer mitochondrial membrane to the inner mitochondrial P-450scc. However, introduction of PBR in the PBR-negative R2C cells rescued steroidogenesis. These results are in agreement with our recent in vivo studies where a drug-induced decrease in adrenal PBR expression resulted in reduced circulating glucocorticoid levels in rats (17Amri H. Ogwuegbu S.O. Boujrad N. Drieu K. Papadopoulos V. Endocrinology. 1996; 137: 5707-5718Crossref PubMed Scopus (106) Google Scholar) and targeted mutagenesis of PBR showed its lethal effect on mouse embryo survival (18Papadopoulos V. Amri H. Boujrad N. Cascio C. Culty M. Garnier M. Hardwick M. Li H. Vidic B. Brown A.S. Reversat J.L. Bernassau J.M. Drieu K. Steroids. 1997; 62: 21-28Crossref PubMed Scopus (335) Google Scholar). Thus, these data provide unequivocal evidence on the determining role of PBR in cholesterol transport and steroid synthesis.Because PBR is a mitochondrial protein (5Papadopoulos V. Endocr. Rev. 1993; 14: 222-240Crossref PubMed Scopus (402) Google Scholar, 7Anholt R.R.H. Pedersen P.L. De Souza E.B. Snyder S.H. J. Biol. Chem. 1986; 261: 576-583Abstract Full Text PDF PubMed Google Scholar), PBR-null cells express high levels of StAR (present study), in vivo reduction of adrenal PBR expression and corticosterone synthesis occurred despite the presence of high levels of StAR (17Amri H. Ogwuegbu S.O. Boujrad N. Drieu K. Papadopoulos V. Endocrinology. 1996; 137: 5707-5718Crossref PubMed Scopus (106) Google Scholar), and StAR does not need to enter the mitochondria to stimulate pregnenolone formation (33Arakane F. Sugawara T. Nishino H. Liu Z. Holt J.A. Pain D. Stocco D.M. Miller W.L. Strauss III, J.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13731-13736Crossref PubMed Scopus (251) Google Scholar), we propose that PBR acts after StAR and that PBR may control or mediate the effect of StAR on mitochondrial steroid synthesis. In addition, the observations that (i) a cytosolic protein is required for the appearance of a higher affinity PBR coupled to constitutive (36Garnier M. Boujrad N. Ogwuegbu S.O. Hudson Jr., J.R. Papadopoulos V. J. Biol. Chem. 1994; 269: 22105-22112Abstract Full Text PDF PubMed Google Scholar) and hormone-induced (52Boujrad N. Gaillard J.-L. Garnier M. Papadopoulos V. Endocrinology. 1994; 135: 1576-1583Crossref PubMed Scopus (56) Google Scholar) steroid synthesis, (ii) PBR is coupled to voltage-dependent anion channel (9Garnier M. Dimchev A. Boujrad N. Price M.J. Musto N.A. Papadopoulos V. Mol. Pharmacol. 1994; 45: 201-211PubMed Google Scholar), a protein located on the outer/inner mitochondrial membrane contact sites, and (iii) the StAR precursor is targeted to mitochondria where it may be cleaved at the contact sites to enter the mitochondria (6Stocco D.M. Clark B.J. Endocr. Rev. 1996; 17: 221-244Crossref PubMed Scopus (926) Google Scholar), suggest that the coordinated interaction of PBR and StAR may be the key to the induction and maintenance of steroid synthesis. We have used the constitutive steroid-producing R2C Leydig cell to construct a PBR-null steroidogenic cell line using a method of gene inactivation based on targeted homologous recombination using selectable marker genes. In this PBR mutant cell line, steroidogenesis ceased due to the arrest of cholesterol transport across the outer mitochondrial membrane to the inner mitochondrial P-450scc. However, introduction of PBR in the PBR-negative R2C cells rescued steroidogenesis. These results are in agreement with our recent in vivo studies where a drug-induced decrease in adrenal PBR expression resulted in reduced circulating glucocorticoid levels in rats (17Amri H. Ogwuegbu S.O. Boujrad N. Drieu K. Papadopoulos V. Endocrinology. 1996; 137: 5707-5718Crossref PubMed Scopus (106) Google Scholar) and targeted mutagenesis of PBR showed its lethal effect on mouse embryo survival (18Papadopoulos V. Amri H. Boujrad N. Cascio C. Culty M. Garnier M. Hardwick M. Li H. Vidic B. Brown A.S. Reversat J.L. Bernassau J.M. Drieu K. Steroids. 1997; 62: 21-28Crossref PubMed Scopus (335) Google Scholar). Thus, these data provide unequivocal evidence on the determining role of PBR in cholesterol transport and steroid synthesis." @default.
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• W2134397312 title "Targeted Disruption of the Peripheral-type Benzodiazepine Receptor Gene Inhibits Steroidogenesis in the R2C Leydig Tumor Cell Line" @default.
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