17Beta-oestradiol up-regulates the expression of a functional UDP-glucose dehydrogenase in articular chondrocytes: comparison with effects of cytokines and growth factors.

OBJECTIVES: To investigate the mechanisms by which cytokines and 17beta-oestradiol (17beta-E2) modulate gene expression and activity of uridine diphosphoglucose dehydrogenase (UGDH), a key enzyme of GAG synthesis in articular chondrocytes. METHODS: Rabbit articular chondrocytes (RAC) from 3-week-old animals were incubated for 24 h with TGF-beta, insulin like growth factor-I (IGF-I), IL-1beta, IL-6 and 17beta-E2. GAG synthesis was measured by [35S]-sulphate labelling and the expression of the UGDH gene was estimated by both real-time polymerase chain reaction and western blotting, whereas the enzyme activity was assayed by a spectrophotometric procedure. In addition, the transcriptional activity of several UGDH gene promoter constructs was determined in RAC transiently transfected with wild-type or deleted human oestrogen receptor-alpha gene (hER alpha66 or hER alpha46, respectively). RESULTS: 17Beta-E2 and its receptor hER alpha66 enhanced GAG neosynthesis in rabbit articular chondrocytes, as did TGF-beta1 whereas IL-1beta decreased this synthesis. 17Beta-E2 was found to exert positive regulatory effects at mRNA, protein and UGDH activity levels. In addition, the receptor hER alpha66, but not hER alpha46, increased the transcriptional activity of the UGDH gene. In contrast, no clear correlation between transcription, translation and activity of the UGDH was found under the effects of the cytokines studied. However, TGF-beta enhanced the enzyme activity, whereas IL-1beta, IL-6 and IGF-I were without significant effect. CONCLUSIONS: 17Beta-E2 enhanced GAG synthesis in chondrocytes via up-regulation of the UGDH gene expression and enzyme activity. These data provide insights into the molecular mechanisms involved in the regulation of the UGDH gene and offer new approaches to investigate its potential alteration in joint diseases.

Antimitotic activity of high affinity ligands for peripheral benzodiazepine receptor (PBR) in some normal and neoplastic cell lines.

PURPOSE: Overexpression of PBR has been found in several tumor types including ovarian, colon, breast adenocarcinomas, esophageal cancer. There is evidence suggesting that PBR ligands regulate cell proliferation. However, their action is probably cell-type specific. We decided to evaluate mitotic activity of PBR ligands in some normal and neoplastic cell lines. MATERIAL AND METHODS: The cells were maintained according to standard procedures. Ligand binding assay was performed in cell extract using PK-11195 or Ro-54864 and [N-methyl-3H] Ro-54864 or [N-methyl-3H] PK-11195. Cell proliferation was evaluated using 5-[3H]-thymidine assay. Western Immunoblot assay was conducted using polyclonal anti-PBR antibody. RESULTS: We have found that, macrophages evoked strong binding of both Ro-54864 and PK-11195. This phenomenon was accompanied by drastic decrease in the cell divisions. Similar effect was found only in the case of non-estrogen-dependent breast cancer cells MDA-MB 231. It suggest that PBR-ligand mediated inhibition of mitogenesis may represent a new anticancer strategy in non-estrogen-dependent breast cancer. In respect to macrophages inhibition of the cell division by both PBR ligands may have implication in modulation of inflammatory response. It has been postulated that PBR ligands may have anti-inflammatory activity in rheumatoid arthritis. The presence of peripheral benzodiazepine receptors in chondrocytes, T cells, macrophages and mesenchymal cells suggest that peripheral benzodiazepine receptor ligands may interfere with the cytokine network and thus modulate inflammatory response. CONCLUSIONS: The data suggest that PBR-ligand mediated inhibition of DNA synthesis in non-estrogen dependent breast cancer cells and in macrophages may represent a new therapeutic approach of breast anti-cancer and anti-inflammatory therapy.

The peroxisome proliferator perfluorodecanoic acid inhibits the peripheral-type benzodiazepine receptor (PBR) expression and hormone-stimulated mitochondrial cholesterol transport and steroid formation in Leydig cells.

The peroxisome proliferator perfluordecanoic acid (PFDA) has been shown to exert an antiandrogenic effect in vivo by acting directly on the interstitial Leydig cells of the testis. The objective of this study was to examine the in vitro effects of PFDA and identify its site of action in steroidogenesis using as model systems the mouse tumor MA-10 and isolated rat Leydig cells. PFDA inhibited in a time- and dose-dependent manner the hCG-stimulated Leydig cell steroidogenesis. This effect was localized at the level of cholesterol transport into the mitochondria. PFDA did not affect either the total cell protein synthesis or the mitochondrial integrity. Moreover, it did not induce any DNA damage. Morphological studies indicated that PFDA induced lipid accumulation in the cells, probably due to the fact that cholesterol mobilized by hCG did not enter the mitochondria to be used for steroidogenesis. In search of the target of PFDA, we examined its effect on key regulatory mechanisms of steroidogenesis. PFDA did not affect the hCG-induced steroidogenic acute regulatory protein (StAR) levels. However, it was found to inhibit the mitochondrial peripheral-type benzodiazepine receptor (PBR) ligand binding capacity, 18-kDa protein, and messenger RNA (mRNA) levels. Further studies indicated that PFDA did not affect PBR transcription, but it rather accelerated PBR mRNA decay. Taken together, these data suggest that PFDA inhibits the Leydig cell steroidogenesis by affecting PBR mRNA stability, thus inhibiting PBR expression, cholesterol transport into the mitochondria, and the subsequent steroid formation. Moreover, this action of PFDA on PBR mRNA stability indicates a new mechanism of action of peroxisome proliferators distinct from the classic transcription-mediated regulation of target genes.

In vitro studies on the role of the peripheral-type benzodiazepine receptor in steroidogenesis.

In vitro studies using isolated cells, mitochondria and submitochondrial fractions demonstrated that in steroid synthesizing cells, the peripheral-type benzodiazepine receptor (PBR) is an outer mitochondrial membrane protein, preferentially located in the outer/inner membrane contact sites, involved in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, the rate-determining step in steroid biosynthesis. Mitochondrial PBR ligand binding characteristics and topography are sensitive to hormone treatment suggesting a role of PBR in the regulation of hormone-mediated steroidogenesis. Targeted disruption of the PBR gene in Leydig cells in vitro resulted in the arrest of cholesterol transport into mitochondria and steroid formation; transfection of the mutant cells with a PBR cDNA rescued steroidogenesis demonstrating an obligatory role for PBR in cholesterol transport. Molecular modeling of PBR suggested that it might function as a channel for cholesterol. This hypothesis was tested in a bacterial system devoid of PBR and cholesterol. Cholesterol uptake and transport by these cells was induced upon PBR expression. Amino acid deletion followed by site-directed mutagenesis studies and expression of mutant PBRs demonstrated the presence in the cytoplasmic carboxy-terminus of the receptor of a cholesterol recognition/interaction amino acid consensus sequence. This amino acid sequence may help for recruiting the cholesterol coming from intracellular sites to the mitochondria.

Acute action of choriogonadotropin on Leydig tumor cells: qualitative and quantitative transformation of lipid moieties.

BACKGROUND: Testicular Leydig cells use either exogenous or de novo synthesized cholesterol as the substrate for the production of testosterone with hormone stimulation. Although the long-term effect of trophic hormones on Leydig cell cholesterol uptake, storage, and deesterification has been well documented, the early effects of the human choriogonadotropin (hCG) on cell cholesterol/lipid distribution are not yet known. METHODS: Sections of cells treated with hCG for 15 sec to 30 min were examined by electron microscopy (EM) for the surface density of lipid moieties in the cytoplasm. In addition, the time-dependent distribution of lipids within the cytoplasmic inclusions and the ultimate destination of this substrate were evaluated by EM. The results were analyzed with standard morphometric methods. RESULTS AND CONCLUSION: The surface density of cytoplasmic lipid pools increased significantly within the 15 sec following the exposure of cells to hCG, and it tapered off to the control level in the subsequent 30 min. Such a fluctuation in the amount of cytoplasmic lipids may be due to (1) the quantity of released substrate from the reticular compartment or (2) the rate of its transport across the outer mitochondrial membrane to the inner mitochondrial cytochrome P450 side-chain cleavage, where steroidogenesis begins with the conversion of cholesterol to pregnenolone. These two processes were not quantitatively coordinated in the stimulated cell during the initial 30 min, resulting in a surplus of cytoplasmic lipid pools. To compensate for such uneven metabolic balance, the cell apparently disposed of the excess substrate by a mechanism of molecular regrouping from a micellar configuration to a bilayer structure followed by exocytosis.

Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis.

Steroidogenesis begins with the metabolism of cholesterol to pregnenolone by the inner mitochondrial membrane cytochrome P450 side-chain cleavage (P450scc) enzyme. The rate of steroid formation, however, depends on the rate of cholesterol transport from intracellular stores to the inner mitochondrial membrane and loading of P450scc with cholesterol. In previous in vitro studies, we demonstrated that a key element in the regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR). We also showed that the polypeptide diazepam binding inhibitor (DBI), an endogenous PBR ligand, stimulates cholesterol transport and promotes loading of cholesterol to P450scc in vitro, and that its presence is vital for hCG-induced steroidogenesis by Leydig cells. Based on these data and the observations that i) the mitochondrial PBR binding and topography are regulated by hormones; ii) the 18-kDa PBR protein is functionally coupled to the mitochondrial contact site voltage-dependent anion channel protein; iii) the 18-kDa PBR protein is a channel for cholesterol, as shown by molecular modeling and in vitro reconstitution studies; iv) targeted disruption of the PBR gene in steroidogenic cells dramatically reduces the ability of the cells to transport cholesterol in the mitochondria and produce steroids; v) endocrine disruptors, with known anisteroidogenic effect, inhibit PBR ligand binding; and vi) in vivo reduction of adrenal PBR expression results in reduced circulating glucocorticoid levels, we conclude that PBR is an indispensable element of the steroidogenic machinery.

Targeted disruption of the peripheral-type benzodiazepine receptor gene inhibits steroidogenesis in the R2C Leydig tumor cell line.

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.

In vivo regulation of peripheral-type benzodiazepine receptor and glucocorticoid synthesis by Ginkgo biloba extract EGb 761 and isolated ginkgolides.

Glucocorticoid excess has broad pathogenic potential including neurotoxicity, neuroendangerment, and immunosuppression. Glucocorticoid synthesis is regulated by ACTH, which acts by accelerating the transport of the precursor cholesterol to the mitochondria where steroidogenesis begins. Ginkgo biloba is one of the most ancient trees, and extracts from its leaves have been used in traditional medicine. A standardized extract of Ginkgo biloba leaves, termed EGb 761 (EGb), has been shown to have neuroprotective and antistress effects. In vivo treatment of rats with EGb, and its bioactive components ginkgolide A and B, specifically reduces the ligand binding capacity, protein, and messenger RNA expression of the adrenocortical mitochondrial peripheral-type benzodiazepine receptor (PBR), a key element in the regulation of cholesterol transport, resulting in decreased corticosteroid synthesis. As expected, the ginkgolide-induced decrease in glucocorticoid levels resulted in increased ACTH release, which in turn induced the expression of the steroidogenic acute regulatory protein. Because ginkgolides reduced the adrenal PBR expression and corticosterone synthesis despite the presence of high levels of steroidogenic acute regulatory protein, these data demonstrate that PBR is indispensable for normal adrenal function. In addition, these results suggest that manipulation of PBR expression could control circulating glucocorticoid levels, and that the antistress and neuroprotective effects of EGb are caused by to its effect on glucocorticoid biosynthesis.

Acute action of choriogonadotropin on Leydig tumor cells: changes in the topography of the mitochondrial peripheral-type benzodiazepine receptor.

We examined the topography of the MA-10 Leydig tumor cell mitochondrial peripheral-type benzodiazepine receptor (PBR). In previous studies, the 18 kDa PBR was found to be functionally associated with the voltage-dependent anion channel, located in the junctions between outer and inner membranes. Transmission electron (TEM) and atomic force microscopy (AFM) of immunogold labeled PBR on Leydig cell mitochondrial preparations showed that the 18 kDa PBR protein is organized in clusters of 4-6 molecules. Addition of hCG to Leydig cells induces a rapid, within 30 sec, increase in PBR ligand binding and morphological changes, namely redistribution of PBR molecules in large clusters (>7 particles). These hCG-induced changes were inhibited by a cAMP-dependent protein kinase inhibitor and by the benzodiazepine flunitrazepam. AFM further demonstrated the rapid reorganization of the mitochondrial membrane, where the formation of contacts between the outer and the inner mitochondrial membrane may facilitate cholesterol transfer.

Evidence for germ cell control of Sertoli cell function in three models of germ cell depletion in adult rat.

In order to clarify the role of germ cells in the regulation of Sertoli cell secretions, three experimental models of germ cell depletion were used: hypodactyl rat mutation (testis weight [TW]: 55% less than controls), in utero busulfan treatment (TW: 88% less than controls), and neonatal experimental cryptorchidism (TW: 72% less than controls). The aim of this work was to compare the numbers of Leydig and Sertoli cells and the production of germ cells in each experimental model to the in vitro secretions of Leydig and Sertoli cells in conditioned media and to the hormonal serum profiles of the same animal in vivo. In the three models, serum levels of hypophyseal and testosterone hormones were significantly increased and decreased, respectively. In the absence of germ cells, the total length of seminiferous tubules, the total numbers of Sertoli and Leydig cells, and the daily production of germ cells were significantly diminished. The addition of both control and damaged seminiferous tubule culture media (STM: media conditioned by 10 cm of seminiferous tubules) to 10(6) control or damaged Leydig cells led to a further increase of testosterone production after ovine LH stimulation. However, expressed per Sertoli cell, testosterone production by control Leydig cells was reduced by addition of damaged STM as compared to addition of control STM, and similarly, the addition of control STM to damaged Leydig cells enhanced testosterone production more than did the addition of damaged STM. Secretions of transferrin per Sertoli cell in STM were reduced as compared to controls by the absence of germ cells but to a lesser extent than was production of spermatocytes and of spermatids. In conclusion, secretions by Sertoli cells of the paracrine factor involved in the control of testosterone production by Leydig cells and of transferrin are modified by germ cells.

Evolution of somatic and germ cell populations after busulfan treatment in utero or neonatal cryptorchidism in the rat.

In order to elucidate the respective effects of depletion of germ cells and of increase in testicular temperature, rats of the same Wistar strain were rendered experimentally bicryptorchid or sterilized by a busulfan injection in utero and compared to control animals. In both models, germ cells were depleted but numeric evolution and functions of somatic cells differed. The aim of that work was to compare the numeric evolutions of testicular somatic and germ cells to their respective functions in each model before puberty and in adult rats of the same strain. Serum concentrations of FSH, LH and testosterone were compared at 20, 40 and 110 days of age. Histological analyses of Sertoli and germ cells in the seminiferous tubules and of Leydig cells in the intertubular tissue were performed before puberty and at adulthood. Testosterone serum concentrations were depleted in both models starting at 40 days of age and more in busulfan-treated rats. Both FSH and LH levels were increased from 20 days onwards in experimental rats. Additional cryptorchidism in busulfan-treated rats depressed the serum testosterone concentration. At 17 days of age, the cryptorchidism do not modify somatic or germ cell populations while busulfan treatment has induced a decrease of both these populations. Conversely, the cross sectional area of the somatic testicular cells was not affected whatever the treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a stimulator of steroid hormone synthesis isolated from testis.

Gonadal steroidogenesis is regulated by pituitary gonadotropins and a locally produced, unidentified factor. A 70-kilodalton (kD) protein complex secreted from rat Sertoli cells was isolated. The complex, composed of 28- and 38-kD proteins, stimulated steroidogenesis by Leydig cells and ovarian granulosa cells in a dose-dependent and adenosine 3',5'-monophosphate-independent manner. The follicle-stimulating hormone-induced 28-kD protein appeared to be responsible for the bioactivity, but the 38-kD protein was indispensable for maximal activity. The 28- and 38-kD proteins were shown to be identical to the tissue inhibitor of metalloproteinase-1 (TIMP-1) and the proenzyme form of cathepsin L, respectively. Thus, a TIMP-1-procathepsin L complex is a potent activator of steroidogenesis and may regulate steroid concentrations and, thus, germ cell development in both males and females.

Acute action of choriogonadotropin on Leydig tumor cells: induction of a higher affinity benzodiazepine-binding site related to steroid biosynthesis.

We previously demonstrated that the mitochondrial peripheral-type benzodiazepine receptor (PBR) is coupled to hormone-activated steroidogenesis by regulating the intramitochondrial cholesterol transport, the rate-determining step of steroid biosynthesis. In the present study we examined whether PBR is the site of hormone action using the hCG-responsive MA-10 mouse Leydig tumor cell line as a model system. Within 15 sec of the addition of hCG to Leydig cells a 3-fold cAMP-dependent increase in PBR binding was observed. This rapid increase returned to basal levels within 60 sec. No effect was observed after 1 min in the continued presence of hCG. Scatchard analysis revealed that in addition to the known high affinity (5.0 nM) benzodiazepine-binding site, a second, hormone-induced, higher affinity (0.2 nM) benzodiazepine-binding site appeared. We then examined whether in such a short time frame steroid synthesis occurs. Fifteen-second incubation of MA-10 cells with the inhibitor of cholesterol metabolism aminoglutethimide together with hCG also resulted in an increased rate of pregnenolone formation by their isolated mitochondria that were washed and incubated in aminoglutethimide-free buffer. The dose response of benzodiazepine binding to hCG closely parallels the increase in steroid formation by the mitochondria of stimulated cells. Addition of the selective inhibitor of cAMP-dependent protein kinase, H-89, completely blocked hormone-induced PBR binding and steroid formation, whereas addition of the inactive analog H-85 was without any effect. The addition of flunitrazepam, a benzodiazepine previously shown to inhibit the trophic hormone action on steroidogenesis, completely abolished the hCG-induced rapid stimulation of steroid synthesis. These results demonstrate that in MA-10 cells, the most rapid effect described thus far of hCG and cAMP, is the transient induction of a higher affinity benzodiazepine-binding site, which occurs concomitantly with an increase in the rate of steroid formation. This, in turn, suggests that these hormones alter PBR to activate cholesterol delivery to the inner mitochondrial membrane and subsequent steroid formation.

The polypeptide diazepam-binding inhibitor and a higher affinity mitochondrial peripheral-type benzodiazepine receptor sustain constitutive steroidogenesis in the R2C Leydig tumor cell line.

The polypeptide diazepam binding inhibitor (DBI) and drug ligands for the mitochondrial peripheral-type benzodiazepine receptor (PBR) have been shown to regulate cholesterol transport, the rate-determining step in steroidogenesis, in hormone-responsive steroidogenic cells including the MA-10 Leydig tumor cells. The present study was designed to characterize the role of DBI and PBR in the R2C rat Leydig tumor constitutive steroid-producing cell model. Both DBI and PBR were present in R2C cells. R2C cell treatment with a cholesterol-linked phosphorothioate oligodeoxynucleotide antisense to DBI, but not sense, resulted in the reduction of DBI levels and a concomitant dramatic decrease of the amount of progesterone produced. These observations strongly suggested that DBI was important in maintaining constitutive steroidogenesis in R2C cells. Radioligand binding assays revealed the presence of a single class of PBR binding sites with an affinity 10 times higher (Kd approximately 0.5 nM) than that displayed by the MA-10 PBR (Kd approximately 5 nM). Photolabeling of R2C and MA-10 cell mitochondria with the photoactivatable PBR ligand [3H]1-(2-fluoro-5-nitrophenyl)-N-methyl-N-(1-methyl-propyl)-3- isoquinolinecarboxamide showed that the M(r) 18,000 PBR protein was specifically labeled. This indicates that the R2C cells express a PBR protein which has properties similar to the MA-10 PBR. Chemical crosslinking studies of purified metabolically radiolabeled DBI to mitochondria provided direct evidence that DBI specifically binds to the M(r) 18,000 PBR protein. Moreover, DBI and a PBR synthetic ligand were able to increase steroid production in isolated R2C cell mitochondria which express the 5 nM affinity receptor. However, mitochondrial PBR binding was increased by 6-fold upon addition of the post-mitochondrial fraction, suggesting that a cytosolic factor modulates the binding properties of PBR in R2C cells and is responsible for the 0.5 nM affinity receptor seen in intact cells. In conclusion, these data demonstrate that DBI plays a key role in maintaining R2C constitutive steroidogenesis by binding to the mitochondrial higher affinity PBR which promotes a continuous supply of cholesterol to the inner mitochondrial side chain cleavage cytochrome P450.

Topography of the Leydig cell mitochondrial peripheral-type benzodiazepine receptor.

Native MA-10 mouse Leydig tumor cell mitochondrial preparations were examined by transmission electron (TEM) and atomic force (AFM) microscopic procedures in order to investigate the topography and organization of the peripheral-type benzodiazepine receptor (PBR). Mitochondria were immunolabeled with an anti-PBR antiserum coupled to gold-labeled secondary antibodies. Results obtained indicate that the 18,000 MW PBR protein is organized in clusters of 4-6 molecules. Moreover, on many occasions, the interrelationship among the PBR molecules was found to favor the formation of a single pore. Taking into account recent observations that the 18,000 MW PBR protein is functionally associated with the pore forming 34,000 MW voltage-dependent anion channel (VDAC) these results suggest that (i) the mitochondrial PBR complex could function as a pore, thus allowing the translocation of cholesterol and other molecules to the inner mitochondrial membrane, and (ii) the native receptor is a multimeric complex of an approximate 140,000 MW composed on an average of five 18,000 PBR subunits, one 34,000 VDAC subunit, and associated lipids.

Studies on the phosphorylation of the 18 kDa mitochondrial benzodiazepine receptor protein.

Steroid biosynthesis activated by pituitary tropic hormones is known to be acutely regulated by cAMP acting via Protein kinase A. Because the mitochondrial benzodiazepine receptor (MBR) has been suggested to play a role in the activation of steroidogenesis, the present study investigates whether various protein kinases phosphorylate MBR. In rat and bovine adrenal mitochondrial preparations Protein kinase A, but not other purified protein kinases, was found to phosphorylate the 18 kDa MBR protein. In digitonin-permeabilized MA-10 Leydig tumor cells incubated with [gamma-32P]ATP, phosphorylation of MBR was detectable during treatment of the cells with dibutyryl cAMP. In conclusion, these data show that the MBR protein is an in vitro and in situ substrate of Protein kinase A, but the role of this phosphorylation in the regulation of steroidogenesis remains to be established.

In vitro reconstitution of a functional peripheral-type benzodiazepine receptor from mouse Leydig tumor cells.

The peripheral-type benzodiazepine receptor (PBR) was identified and characterized by its high affinity for two distinct classes of compounds, the benzodiazepines (BZs) and the isoquinolines (IQs). An M(r) 18,000 IQ-binding protein has been identified as the PBR. In this report we isolated and sequenced a 626-base pair cDNA, specifying an open reading frame of 169 amino acid residues with a predicted molecular weight of 18,843, from MA-10 mouse tumor Leydig cells [i.e., mouse peripheral-type benzodiazepine receptor (mPBR)]. Expression of mPBR cDNA in simian virus 40-transformed 3T3 fibroblasts resulted in an increase in the density of both BZ and IQ binding sites. To examine whether the increased drug binding was due to the M(r) 18,000 PBR protein alone or to other constitutively expressed components of the receptor, an in vitro system was developed using recombinant mPBR protein. The mPBR cDNA was inserted in the pMAL-c2 vector downstream from the malE gene, which encodes maltose-binding protein (MBP). Transfection of the recombinant pMAL-c2 in Escherichia coli provided high levels of expression of the MBP-mPBR fusion protein. Purified MBP-mPBR recombinant fusion protein incorporated into liposomes, but not MBP alone, was able to bind IQs but not BZs. Addition of MA-10 mitochondrial extracts to the liposomes resulted in the restoration of BZ binding. The protein responsible for this effect was then purified and identified as the M(r) 34,000 voltage-dependent anion channel protein, which by itself does not express any BZ and IQ binding. These results provide strong evidence that PBR is not a single protein receptor but a multimeric complex in which the IQ binding site is on the M(r) 18,000 subunit and expression of the BZ binding site requires both the M(r) 18,000 and 34,000 voltage-dependent anion channel subunits.

Inhibition of hormone-stimulated steroidogenesis in cultured Leydig tumor cells by a cholesterol-linked phosphorothioate oligodeoxynucleotide antisense to diazepam-binding inhibitor.

The polypeptide diazepam-binding inhibitor (DBI) has been previously shown to stimulate testicular Leydig, adrenocortical, and glial-cell mitochondrial steroidogenesis in vitro. To assess the in situ role of DBI in trophic hormone-stimulated steroidogenesis, we suppressed DBI levels in the hormone-responsive MA-10 Leydig tumor cells, using a cholesterol-linked phosphorothioate oligodeoxynucleotide (Chol-odN) antisense to DBI. Treating MA-10 cells with Chol-odN antisense to DBI resulted in a dose-dependent reduction of DBI levels (ED50 = 1 microM). In contrast, Chol-odN sense to DBI did not affect its expression. Saturating amounts of human choriogonadotropin (hCG) increased MA-10 progesterone production by 150-fold. Addition of increased concentrations of Chol-odNs sense to DBI or of a nonrelated sequence did not reduce the MA-10 response to hCG. However, in the presence of Chol-odN antisense to DBI that could reduce DBI levels, MA-10 cells lost their ability to respond to hCG (ED50 = 1 microM). In these studies the hCG-stimulated cAMP levels and cytochrome P450 side-chain cleavage activity, as measured by metabolism of 22(R)-hydroxycholesterol, were not affected by the Chol-odNs used. These observations provide unequivocal evidence that DBI plays a vital role in the acute stimulation of steroidogenesis by trophic hormones.