Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis.

Congenital lipoid adrenal hyperplasia is an autosomal recessive disorder that is characterized by impaired synthesis of all adrenal and gonadal steroid hormones. In three unrelated individuals with this disorder, steroidogenic acute regulatory protein, which enhances the mitochondrial conversion of cholesterol into pregnenolone, was mutated and nonfunctional, providing genetic evidence that this protein is indispensable normal adrenal and gonadal steroidogenesis.

Expression of the steroidogenic acute regulatory (StAR) protein: a novel LH-induced mitochondrial protein required for the acute regulation of steroidogenesis in mouse Leydig tumor cells.

The acute response of steroidogenic cells to hormone stimulation is the mobilization of cholesterol from cellular stores and the outer mitochondrial membrane to the inner mitochondrial membrane and the cholesterol side-chain cleavage complex (CSCC) where the first enzymatic reaction occurs. It has been well established that the translocation of cholesterol from the outer to the inner mitochondrial membrane requires de novo protein synthesis and that this process is the rate-limiting, regulated step in steroidogenesis. We have purified a novel mitochondrial protein (named StAR) from the MA-10 mouse Leydig tumor cells which we have previously proposed represents a strong candidate for the newly synthesized regulatory protein in steroidogenesis.

The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR).

The acute response of steroidogenic cells to trophic hormone stimulation is the mobilization of cholesterol from cellular stores to the mitochondrial outer membrane and the transfer of this cholesterol to the mitochondrial inner membrane where the first enzymatic step in steroidogenesis occurs. The transfer of cholesterol across the mitochondrial membranes is dependent upon de novo protein synthesis, and this is the regulated step in the process. Although the newly synthesized regulatory protein(s) have yet to be identified, we previously have proposed a candidate protein which we identified in MA-10 cells that is synthesized in response to luteinizing hormone stimulation and that is localized to the mitochondria.

Is ouabain an authentic endogenous mammalian substance derived from the adrenal?

Ouabain has recently been reported to be an endogenous mammalian substance released by the adrenal cortex and present in normal plasma. We have attempted to confirm and extend this observation. Using a ouabain radioimmunoassay developed in this laboratory, we fractionated by high-performance liquid chromatography (HPLC) normal human plasma from healthy volunteers to determine the presence of ouabain immunoreactivity and compare this immunoreactivity with authentic ouabain.

Report on a 3-year follow-up zidovudine (AZT) treatment in a group of HIV-positive patients with congenital clotting disorders.

Eleven (50%) of 22 HIV-seropositive patients suffering from congenital coagulation defects and followed at the Hemophilia Center of Padua met the eligibility criteria for zidovudine (AZT) therapy. A 3-year clinical and laboratory follow up is described. Mean length of AZT treatment was 14.

The effects of hydrogen peroxide on steroidogenesis in mouse Leydig tumor cells.

It has previously been reported that treatment of rat luteal cells and human granulosa luteal cells with hydrogen peroxide (H2O2) results in a significant inhibition of steroid production. The mechanism of inhibition in the former was found to be at the level of cholesterol transport into the mitochondria, whereas in the latter it was found to be a result of inhibition of one or more enzymes in the steroidogenic pathway. In the present study we examined the effects of H2O2 on hormone-stimulated steroid production in another steroidogenic cell type, the Leydig cell.

The requirement of phosphorylation on a threonine residue in the acute regulation of steroidogenesis in MA-10 mouse Leydig cells.

In the present study we have used several non-phosphorylatable analogs of the amino acids threonine and serine to determine the role of phosphorylation in the acute regulation of steroidogenesis in MA-10 mouse Leydig tumor cells. Our results indicate that substitution of the threonine analog into protein results in a inhibition of hormone stimulated steroid production in these cells while none of the serine analogs employed displayed a similar inhibition. Strikingly, only the threonine analog resulted in the inhibition of the synthesis of several 30 kDa mitochondrial proteins which we have previously shown to be induced by hormone stimulation of MA-10 cells.

The use of genetic manipulation of MA-10 Leydig tumor cells to demonstrate the role of mitochondrial proteins in the acute regulation of steroidogenesis.

The true rate-limiting step in steroidogenesis is the delivery of cholesterol to the inner mitochondrial membrane where it is converted to pregnenolone by the cholesterol side-chain cleavage complex. This process is known to require de novo protein synthesis. We have previously described the synthesis of a family of 37, 32, and 30 kilodalton mitochondrial proteins in response to hormone stimulation in MA-10 mouse Leydig tumor cells and have proposed that these proteins are involved in the acute regulation of steroidogenesis.

Bleeding as a cause of death in HIV-seropositive patients with congenital clotting disorders.

The causes of death in a group of HIV-seropositive patients suffering from congenital clotting disorders (cCD) were studied. During the past 6 years, we have followed 19 patients with cCD and HIV infection. Eight patients fulfilled revised CDC criteria for AIDS, 6 subjects reached stage III of CDC, and 5 remained asymptomatic (CDC stage II).

Further evidence that the mitochondrial proteins induced by hormone stimulation in MA-10 mouse Leydig tumor cells are involved in the acute regulation of steroidogenesis.

In previous studies we and others have described several mitochondrial proteins which are synthesized in response to acute hormone stimulation in several steroidogenic tissues. In both MA-10 mouse Leydig tumor cells and primary cultures of rat adrenal cortex cells, these proteins consist of a family of 37 kilodalton (kDa) and 32 kDa precursor forms and fully processed forms which are 30 kDa in molecular weight. The nature of the appearance of these proteins and their subcellular localization to the mitochondria, the site of the rate limiting step in steroidogenesis, has led to the speculation that they may be involved in the acute regulation of steroidogenesis.

Effects of steroidogenesis inducing protein (SIP) on steroid production in MA-10 mouse Leydig tumor cells: utilization of a non-cAMP second messenger pathway.

We have investigated the effects of steroidogenesis inducing protein (SIP) (Endocrinology (1990) 126, 3043-3052) on steroid production in MA-10 mouse Leydig tumor cells. Our results indicate that SIP results in the stimulation of progesterone production in MA-10 cells to the same extent obtained when maximal doses of luteinizing hormone (LH), human chorionic gonadotropin (hCG) and dibutyryl cAMP (dbcAMP) are used. It was also observed that the increased progesterone production in response to SIP was not accompanied by an increase in intracellular cAMP levels as was seen following hCG stimulation.

Sterol carrier protein 2 (non-specific lipid transfer protein) is localized in membranous fractions of Leydig cells and Sertoli cells but not in germ cells.

The cellular and subcellular distribution of sterol carrier protein 2 (SCP2; nsL-TP) was reinvestigated in rat testicular cells by Western blotting and immunocytochemistry, using the affinity purified antibody against rat liver SCP2. Western blot analysis revealed high levels of the protein in the somatic cells of the testis, e.g.

The 30-kDa mitochondrial proteins induced by hormone stimulation in MA-10 mouse Leydig tumor cells are processed from larger precursors.

Acute regulation of steroidogenesis in steroidogenic tissue is controlled by the transfer of cholesterol from the outer to the inner mitochondrial membrane where cleavage to produce pregnenolone occurs. Hormonal stimulation of MA-10 mouse Leydig tumor cells results in a large increase in steroidogenesis and the concomitant appearance of a series of 30-kDa proteins which have been localized to the mitochondria. In the present study we have shown that the appearance of these proteins occurs in a dose-responsive manner with both human chorionic gonadotropin and cyclic AMP analog.

Effect of different steroidogenic stimuli on protein phosphorylation and steroidogenesis in MA-10 mouse Leydig tumor cells.

Numerous studies have indicated that treatment of Leydig cells with gonadotropin results in increased levels of intracellular cAMP, binding of cAMP to and activation of protein kinase A, phosphorylation of proteins, synthesis of new proteins and eventually, stimulation of steroidogenesis. In addition, recent studies have indicated that protein phosphorylation is an indispensable event in the production of steroids in response to hormone stimulation in adrenal cells. Because of the important role of phosphorylation in steroidogenic regulation, we investigated the effects of human chorionic gonadotropin (hCG), dibutyryl cyclic AMP (dbcAMP), forskolin and the phorbol ester, phorbol-12-myristate 13-acetate (PMA) on protein phosphorylation in MA-10 mouse Leydig tumor cells.

Presence of identical mitochondrial proteins in unstimulated constitutive steroid-producing R2C rat Leydig tumor and stimulated nonconstitutive steroid-producing MA-10 mouse Leydig tumor cells.

The acute regulation of steroidogenesis in steroidogenic tissues requires de novo protein synthesis. It is believed that these newly synthesized proteins are instrumental in the delivery of the substrate, cholesterol, to the inner mitochondrial membrane where the cholesterol side-chain cleavage complex converts cholesterol to pregnenolone. A number of studies have attempted to characterize the protein(s) synthesized in response to hormone stimulation and, hence, function in the delivery of cholesterol to the cholesterol side-chain cleavage complex.

Effects of hypophysectomy and human chorionic gonadotrophin on Leydig cell function in mature rats.

The biochemical activities involved in the maintenance of Leydig cell functions, and the effects of hypophysectomy and human chorionic gonadotrophin (hCG) on these functions are largely unknown. In the present study, adult hypophysectomized rats were used as a model to determine the effects of these treatments on a number of biochemical and morphological parameters. After 33 days of hypophysectomy, the morphology of the Leydig cells had been drastically altered.

An in vitro cell model system to study the action of retinoids on Leydig cell steroidogenesis.

In this study we examined the effects of retinol and retinoic acid on steroid production in MA-10 mouse Leydig tumor cells. Results showed that both retinol and retinoic acid greatly increased progesterone production in this cloned cell line. The stimulatory effect of retinoids is not inhibited by cycloheximide suggesting that de novo protein synthesis is not required.

Regulation of steroidogenesis in subclones of the MA-10 mouse Leydig tumor cell line.

We have previously reported the isolation of a subclone of the MA-10 mouse Leydig tumor cell line (MA-10 LP) which secretes less than 10% of the steroid synthesized by the parent, accumulates comparable amounts of cAMP and has equivalent cholesterol side-chain cleavage activity as the parent population (Kilgore and Stocco (1989) Endocrinology 124, 1210-1216). In the present study we show that addition of exogenous sterol carrier protein 2 (SCP2) to isolated mitochondria was not able to overcome the deficient steroid response of MA-10 LP. We have also demonstrated that human chorionic gonadotropin (hCG)-stimulated cellular events which activate steroid production by subsequently isolated mitochondria require ongoing protein synthesis, release of intracellular calcium and are mediated through the calcium-calmodulin complex.

Evidence for the functional coupling of cyclic AMP in MA-10 mouse Leydig tumour cells.

A number of studies have indicated that increased production of steroids can be obtained with doses of tropic hormone which do not result in detectable increases in intracellular cAMP. It has been suggested that this may be a result of compartmentalization or functional coupling of cAMP generated by hormone-receptor interactions to specific steroid producing pathways in the cell. In the present study we have stimulated the MA-10 mouse Leydig tumour cell with hCG, dibutyryl cAMP (dbcAMP) and forskolin to determine if functional coupling of cAMP occurs.

An endogenous digitalis-factor derived from the adrenal gland: studies of adrenocortical tumor cells.

The present studies demonstrate that the murine adrenocortical tumor cell line Y-1 releases a digoxin-like immunoreactive material into both serum-supplemented nutrient medium and minimal Krebs-Ringer bicarbonate medium. Release of pregnenolone into minimal medium from these cells was consistently inhibited by addition of the cholesterol side-chain cleavage inhibitor aminoglutethimide. However, release of digoxin-like immunoreactivity (DLI) was not similarly affected.

An endogenous digitalis-like factor derived from the adrenal gland: studies of adrenal tissue from various sources.

We have previously reported that the adrenal gland is the probable origin of the digitalis-like immunoreactive material (DLI) present in the plasma of rats and other species which have never received cardiac glycoside drugs. The present study demonstrates that adrenal glands removed from rats and then chopped release an immunoreactive digitalis-like material into a serum-free minimal incubation medium. HPLC studies indicated that this immunoreactivity was not homogeneous.

Comparison of cellular and secreted proteins of macrophages from the testis and peritoneum on two-dimensional polyacrylamide gels: evidence of tissue specific function.

It has been shown that the testis contains a population of cells with many characteristics typical of macrophages of other tissues. However, these macrophages are unique in that they secrete a product(s) that is responsible for stimulating testosterone secretion by Leydig cells while peritoneal macrophages have no similar effect. The purpose of the present study was to compare the pattern of cellular and secreted proteins of rat testicular macrophages to those of peritoneal macrophages using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).

Initial characterization of a subclone of the MA-10 mouse Leydig tumor cell line.

Cloned cell lines have proven to be useful models in understanding the regulation of endocrine cells and steroid synthesis. In this study we report the isolation and characterization of a subclone of the MA-10 Leydig tumor cell line. Whereas there was no difference in basal steroid production between the clone (MA-10 LP) and the parent stock (MA-10), MA-10 LP produces very low levels of progesterone after stimulation by hCG or (Bu)2cAMP.

Effect of retinol and retinoic acid on testosterone production by rat Leydig cells in primary culture.

Adult rat Leydig cells, purified by Percoll density gradient centrifugation, were used to determine the effect of retinol and retinoic acid on steroidogenesis. It was found that both retinoic acid and retinol stimulated testosterone production. Although retinol was less potent than retinoic acid, retinol had the greater efficacy.

Inhibition of hCG- and cAMP-stimulated progesterone production in MA-10 mouse Leydig tumor cells by ketoconazole.

In this study we attempted to examine the effects of ketoconazole on steroid biosynthesis and to determine which steps in the steroidogenic pathway were blocked using MA-10 Mouse Leydig tumor cells. This cloned cell line produces progesterone as the major steroid following stimulation by hCG or dbcAMP. At a concentration of 1 microM ketoconazole completely inhibited the hCG- and dbcAMP-stimulated progesterone synthesis in MA-10 Leydig cells.