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D M Stocco

Publications and source records attributed to D M Stocco.

At least 37 records · Page 2Linked to original sources
PubMed2000

The role of arachidonic acid in steroidogenesis and steroidogenic acute regulatory (StAR) gene and protein expression.

This study was conducted to examine the mechanism for arachidonic acid (AA) regulation of steroidogenic acute regulatory (StAR) protein expression and the relationship between AA and cAMP in hormone-induced steroidogenesis. Dibutyryl cyclic AMP (Bt(2)cAMP)-stimulated MA-10 Leydig cells were treated with AA and/or the phospholipase A(2) inhibitor, dexamethasone. Dexamethasone significantly reduced Bt(2)cAMP-stimulated progesterone production, StAR promoter activity, StAR mRNA, and StAR protein. The inhibitory effects of dexamethasone were reversed by the addition of 150 microm AA to MA-10 cells. In addition, MA-10 cells were treated with the lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), the 5-lipoxygenase inhibitor, AA861, the epoxygenase inhibitor, miconazole, and the cyclooxygenase inhibitor, indomethacin. Both NDGA and AA861 inhibited progesterone production and StAR protein expression. AA861-inhibited progesterone synthesis and StAR protein were partially reversed by addition of the 5- lipoxygenase metabolite, 5(S)-hydroperoxy-(6E,8Z,11Z, 14Z)-eicosatetraenoic acid. Inhibition of epoxygenase activity inhibited progesterone production significantly, but StAR protein was only slightly reduced. Indomethacin enhanced StAR protein expression and significantly increased progesterone production. Inhibition of AA release or lipoxygenase activities did not affect protein kinase A activity, whereas inhibition of protein kinase A activity using H89 reduced Bt(2)cAMP-induced StAR protein. AA alone did not induce StAR protein expression nor steroid production. These results demonstrate the essential role of AA in steroid biosynthesis and StAR gene transcription and suggest the possible involvement of the lipoxygenase pathway in steroidogenesis. This study further indicates that AA and cAMP transduce signals from trophic hormone receptors to the nucleus through two separate pathways and act to co-regulate steroid production and StAR gene expression and indicates that both pathways are required for trophic hormone-stimulated steroidogenesis.

Animals↗
PubMed2000

Intramitochondrial cholesterol transfer.

Cholesterol serves as the initial substrate for all steroid hormones synthesized in the body regardless of the steroidogenic tissue or final steroid produced. The first steroid formed in the steroidogenic pathway is pregnenolone which is formed by the excision of a six carbon unit from cholesterol by the cytochrome P450 side chain cleavage enzyme system which is located in the inner mitochondrial membrane. It has long been known that the regulated biosynthesis of steroids is controlled by a cycloheximide sensitive factor whose function is to transfer cholesterol from the outer to the inner mitochondrial membrane, thus, the identity of this factor is of great importance. A candidate for the regulatory factor is the mitochondrial protein, the steroidogenic acute regulatory (StAR) protein. Cloning and sequencing of the StAR cDNA indicated that it was a novel protein, and transient transfections with the cDNA for the StAR protein resulted in increased steroid production in the absence of stimulation. Mutations in the StAR gene cause the potentially lethal disease congenital lipoid adrenal hyperplasia, a condition in which cholesterol transfer to the cytochrome P450 side chain cleavage enzyme, P450scc, is blocked, filling the cell with cholesterol and cholesterol esters. StAR knockout mice have a phenotype which is essentially identical to the human condition. The cholesterol transferring activity of StAR has been shown to reside in the C-terminal part of the molecule and a protein sharing homology with a region in the C-terminus of StAR has been shown to display cholesterol transferring capacity. Recent evidence has indicated that StAR can act as a sterol transfer protein and it is perhaps this characteristic which allows it to mobilize cholesterol to the inner mitochondrial membrane. However, while it appears that StAR is the acute regulator of steroid biosynthesis via its cholesterol transferring activity, its mechanism of action remains unknown.

Adrenal Cortex Hormones↗
PubMed2000

Generation of stable cell lines by spontaneous immortalization of primary cultures of porcine granulosa cells.

We report the generation of stable cell lines obtained by spontaneous immortalization of primary cultures of porcine granulosa cells. Three hundred stable cell lines were obtained from three independent immortalization trials. Two of these cell lines retained the steroidogenic capabilities characteristic of granulosa cells, such as de novo synthesis of progesterone and conversion of androstenedione into estradiol-17beta. All the stable cell lines expressed the P450arom and 3betaHSD genes, confirming their granulosa origin. Moreover, the steroidogenic stable granulosa cells also expressed StAR and P450scc genes. Stable cells were developed in cultures using Medium 199 supplemented with 5% newborn calf serum (NBCS). The surviving cells overcame the senescent phase and entered a stage of continuous growth for over one hundred generations. No stable colonies were obtained from cultures grown in MEM or DMEM or media supplemented with 10% NBCS or 5 and 10% fetal calf serum (FCS). Medium 199 is a formulation richer in nutrients compared to MEM or DMEM and the cell growth capability of NBCS is lower than that of FCS, probably due to deficiency of growth factors. We speculate that spontaneous immortalization of granulosa cells may be facilitated by using a rich culture formulation supplemented with low concentrations of serum deficient in growth factors. We have validated the stable cell lines for studying the effect of hormonal steroids on granulosa cell steroidogenesis and the expression of the steroidogenic genes. Therefore, we believe that they are useful models to study the molecular mechanism involved in granulosa cell differentiation and steroidogenesis.

Animals↗
PubMed2000

Effects of lindane on steroidogenesis and steroidogenic acute regulatory protein expression.

Lindane, the gamma isomer of hexachlorocyclohexane (HCH), is one of the oldest synthetic pesticides still in use worldwide. Numerous reports have shown that this pesticide adversely affects reproductive function in animals. Although the pathogenesis of reproductive dysfunction is not yet fully understood, recent reports indicate that lindane can directly inhibit adrenal and gonadal steroidogenesis. Because Leydig cells play a pivotal role in male reproductive function through the production of testosterone, the mouse MA-10 Leydig tumor cell line was used to assess the potential effects of gamma-HCH and its isomers, alpha-HCH and delta-HCH, on steroid production, steroidogenic enzyme expression and activity, and steroidogenic acute regulatory (StAR) protein expression. StAR mediates the rate-limiting and acutely regulated step in hormone-stimulated steroidogenesis, the intramitochondrial transfer of cholesterol to the P450(scc) enzyme. Our studies demonstrate that alpha-, delta-, and gamma-HCH inhibited dibutyryl ([Bu](2)) cAMP-stimulated progesterone production in MA-10 cells in a dosage-dependent manner without affecting general protein synthesis; and protein kinase A or steroidogenic enzyme expression, activity, or both. In contrast, each of these isomers dramatically reduced (Bu)(2)cAMP-stimulated StAR protein levels. Therefore, our results are consistent with the hypothesis that alpha-, delta-, and gamma-HCH inhibited steroidogenesis by reducing StAR protein expression, an action that may contribute to the pathogenesis of lindane-induced reproductive dysfunction.

3-Hydroxysteroid Dehydrogenases↗
PubMed2000

Mammalian bufadienolide is synthesized from cholesterol in the adrenal cortex by a pathway that Is independent of cholesterol side-chain cleavage.

An increasing body of evidence suggests that an endogenous mammalian bufadienolide (BD) may be involved in the regulation of Na(+),K(+)-ATPase activity and the pathogenesis of arterial hypertension. We developed a purification scheme for marinobufagenin (MBG), an amphibian cardiotonic BD, and applied it to purify and characterize material in human plasma, culture medium conditioned by Y-1 adrenocortical cells, and rat adrenal tissue. MBG immunoreactivity purified from plasma and measured by ELISA showed important similarities (chromatography and antibody cross-reactivity) to material secreted into cell culture medium by Y-1 cells. This observation indicates that circulating mammalian BD may have an adrenocortical origin. Release of mammalian BD from adrenocortical cells grown in the absence of exogenous cholesterol was reduced by treatment of cultures with mevastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. Supplementation of the serum and cholesterol-free cell culture medium with the LDL fraction of human plasma increased the production of MBG material in the presence of mevastatin, supporting its origin from cholesterol. We used Y-1 cell lines transfected with genes shown to inhibit steroidogenesis through cholesterol side-chain cleavage (Y-1/DAX and Y-1/RIAB) to investigate the dependence of MBG biosynthesis on side-chain cleavage. Our results indicate that the mammalian BD is synthesized in the adrenal cortex from cholesterol and shares important similarities with the amphibian BD MBG, that its biosynthesis is independent of transfer of cholesterol to the side-chain cleavage enzyme complex mediated by steroidogenic acute regulatory protein, and that neither cAMP nor protein kinase A appears to be a critical component of the pathway controlling its biosynthesis.

Adrenal Cortex↗
PubMed2000

Regulation by adrenocorticotropin (ACTH), angiotensin II, transforming growth factor-beta, and insulin-like growth factor I of bovine adrenal cell steroidogenic capacity and expression of ACTH receptor, steroidogenic acute regulatory protein, cytochrome P450c17, and 3beta-hydroxysteroid dehydrogenase.

The purpose of this study was to evaluate the time-course effect of a 36-h treatment with ACTH (10(-8) M), transforming growth factor-beta1 (TGFbeta1; 10(-10) M), angiotensin II (AngII; 10 (-7) M), and insulin-like growth factor I (IGF-I; 10(-8) M) on the steroidogenic capacity of bovine adrenocortical cells (BAC) and on messenger RNA (mRNA) levels of ACTH receptor, cytochrome P450c17, 3beta-hydroxysteroid dehydrogenase (3betaHSD), steroidogenic acute regulatory protein (StAR), and StAR protein. ACTH and IGF-I enhanced, in a time-dependent manner, the acute 2-h ACTH-induced cortisol production, whereas TGFbeta 1 and AngII markedly reduced it. ACTH, IGF-I, and AngII increased ACTH receptor mRNA, but the opposite was observed after TGFbeta1 treatment. ACTH and IGF-I increased P450c17 and 3betaHSD mRNAs, whereas AngII and TGFbeta1 had the opposite effects. However, the effects of the four peptides on ACTH-induced cortisol production appeared before any significant alterations of the mRNA levels occurred. The most marked and rapid effect of the four peptides was on StAR mRNA. The stimulatory effect of ACTH was seen within 1.5 h, peaked at 4-6 h, and declined thereafter, but at the end of the 36-h pretreatment, the levels of StAR mRNA and protein were higher than those in control cells. IGF-I also enhanced StAR mRNA levels within 1.5 h, and these levels remained fairly constant. The effects of AngII on StAR mRNA expression were biphasic, with a peak within 1.5-3 h, followed by a rapid decline to almost undetectable levels of both mRNA and protein. TGFbeta1 had no significant effect during the first 3 h, but thereafter StAR mRNA declined, and at the end of the experiment the StAR mRNA and protein were almost undetectable. Similar results were observed when cells were treated with ACTH plus TGFbeta1. A 2-h acute ACTH stimulation at the end of the 36-h pretreatment caused a higher increase in StAR mRNA and protein in ACTH- or IGF-I-pretreated cells than in control cells, which, in turn, had higher levels than cells pretreated with TGFbeta1, ACTH plus TGFbeta1, or AngII. These results and the fact that the stimulatory (IGF-I) or inhibitory (AngII and TGFbeta1) effects on ACTH-induced cortisol production were more pronounced than those on the ability of cells to transform pregnenolone into cortisol strongly suggest that regulation of StAR expression is one of the main factors, but not the only one, involved in the positive (IGF-I) or negative (TGFbeta1 and AngII) regulation of BAC for ACTH steroidogenic responsiveness. A high correlation between steady state mRNA level and acute ACTH-induced cortisol production favors this conclusion.

3-Hydroxysteroid Dehydrogenases↗
PubMed2000

Roundup inhibits steroidogenesis by disrupting steroidogenic acute regulatory (StAR) protein expression.

Recent reports demonstrate that many currently used pesticides have the capacity to disrupt reproductive function in animals. Although this reproductive dysfunction is typically characterized by alterations in serum steroid hormone levels, disruptions in spermatogenesis, and loss of fertility, the mechanisms involved in pesticide-induced infertility remain unclear. Because testicular Leydig cells play a crucial role in male reproductive function by producing testosterone, we used the mouse MA-10 Leydig tumor cell line to study the molecular events involved in pesticide-induced alterations in steroid hormone biosynthesis. We previously showed that the organochlorine insecticide lindane and the organophosphate insecticide Dimethoate directly inhibit steroidogenesis in Leydig cells by disrupting expression of the steroidogenic acute regulatory (StAR) protein. StAR protein mediates the rate-limiting and acutely regulated step in steroidogenesis, the transfer of cholesterol from the outer to the inner mitochondrial membrane where the cytochrome P450 side chain cleavage (P450scc) enzyme initiates the synthesis of all steroid hormones. In the present study, we screened eight currently used pesticide formulations for their ability to inhibit steroidogenesis, concentrating on their effects on StAR expression in MA-10 cells. In addition, we determined the effects of these compounds on the levels and activities of the P450scc enzyme (which converts cholesterol to pregnenolone) and the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzyme (which converts pregnenolone to progesterone). Of the pesticides screened, only the pesticide Roundup inhibited dibutyryl [(Bu)(2)]cAMP-stimulated progesterone production in MA-10 cells without causing cellular toxicity. Roundup inhibited steroidogenesis by disrupting StAR protein expression, further demonstrating the susceptibility of StAR to environmental pollutants.

Animals↗
PubMed2000

The role of the StAR protein in steroidogenesis: challenges for the future.

The steroidogenic acute regulatory or StAR protein has been shown to be instrumental in the acute regulation of steroid hormone biosynthesis through its action in mediating cholesterol transfer to the inner mitochondrial membrane and the cholesterol side chain cleavage enzyme system. Since the time of its cloning in 1994, a number of studies have been performed which underscore the important role that this protein plays in steroidogenesis. While it is now quite apparent that StAR fulfills the criteria for the acute regulator as proposed by early studies, several crucial areas remain poorly understood. This list is topped by the so far intractable nature of the mechanism of action of StAR in transferring cholesterol to the P450scc enzyme. A second area which should prove to be of great interest is that of further understanding the regulation of the StAR gene which, like many genes, is quite complex. Lastly, with the recent demonstration of StAR being present in the brain, determining if StAR has a role in the synthesis of neurosteroids should prove to be of great importance.

Adrenal Glands↗
PubMed2000

Dimethoate inhibits steroidogenesis by disrupting transcription of the steroidogenic acute regulatory (StAR) gene.

Dimethoate is a widely used organophosphate insecticide that has been shown to disrupt reproductive function in animals. Although the pathogenesis of Dimethoate-induced reproductive toxicity remains to be determined, a reduction in serum testosterone levels is thought to play an important role in the development of Dimethoate-induced infertility. Since Leydig cells play a crucial role in male reproductive function by producing testosterone, the mouse MA-10 Leydig tumor cell line was used to determine if Dimethoate can directly block steroid hormone biosynthesis and to identify the site of steroidogenic inhibition. Dimethoate inhibited steroidogenesis in both a dose- and time-dependent manner without affecting total protein synthesis or protein kinase A activity. While it decreased the activity of the P450 side chain cleavage (P450 scc) enzyme, a reduction in the activity of this enzyme alone could not account for the level of Bu(2)cAMP-inhibited progesterone production. Instead, our results suggest that Dimethoate inhibited steroidogenesis primarily by blocking transcription of the steroidogenic acute regulatory (StAR) gene. This finding is significant since StAR protein mediates the rate-limiting and acutely-regulated step in steroidogenesis, the transfer of cholesterol from the outer to the inner mitochondrial membrane. This study indicates that StAR may be an important target for environmental pollutants which disrupt steroidogenesis and impair reproductive function.

3-Hydroxysteroid Dehydrogenases↗
PubMed1999

Cyclic AMP and arachidonic acid: a tale of two pathways.

Increasing evidence in recent years has demonstrated the regulatory effects of arachidonic acid and its metabolites on steroid hormone production in various steroidogenic tissues. In trophic hormone-stimulated steroidogenesis, arachidonic acid is rapidly released from phospholipids. This release is dependent upon hormone-receptor interaction and inhibition of arachidonic acid release results in an inhibition of steroidogenesis. Several of the earlier studies indicated that arachidonic acid acts at the rate-limiting step of steroid biosynthesis, the transfer of substrate cholesterol to the inner mitochondrial membrane, but the manner in which this occurred was not clear. Recently it has been demonstrated that arachidonic acid release can participate in the regulation of gene expression of the steroidogenic acute regulatory (StAR) protein which mediates cholesterol transfer to the inner mitochondrial membrane. These studies suggest that this fatty acid may be instrumental in transducing a signal from trophic hormone/receptor interaction to the nucleus utilizing a pathway different from the reported cyclic AMP pathway. It is possible that these two pathways cooperate and serve to co-regulate transcription factors, resulting in StAR gene expression and subsequent steroid production. This hypothesis may serve to explain and co-ordinate previous observations on the roles of cyclic AMP (cAMP) and arachidonic acid in steroid hormone biosynthesis.

Animals↗
PubMed1999

Pregnenolone synthesis in immature rat Sertoli cells.

It has been reported that testicular Sertoli cells can be induced to synthesize the steroidogenic acute regulatory (StAR) protein. StAR mediates the rate-limiting step of steroidogenesis, which is the transfer of cholesterol to the inner mitochondrial membrane. Since Sertoli cells are thought to be unable to utilize cholesterol for the synthesis of steroids the role of StAR in these cells was questioned. In the present studies we have corroborated the induction of StAR protein in immature cultured Sertoli cells in response to either trophic hormone or cAMP analog stimulation. Further, we have shown that long term stimulation of Sertoli cells with cAMP analog results in the induction of P450scc enzyme and increased pregnenolone production. In this manner, the Sertoli cell may resemble its ovarian homolog, the granulosa cell, more closely than previously thought with regards to its steroidogenic capacity. Thus, StAR may play the same role in Sertoli cells as it does in other steroidogenic tissues.

Animals↗
PubMed1999

Transcriptional regulation of the StAR gene.

The steroidogenic acute regulatory (StAR) protein regulates the rate-limiting step of steroidogenesis. In steroidogenic tissues, the StAR gene is regulated acutely by trophic hormone through a cAMP second messenger pathway. Thus, the gene encoding StAR must be finely regulated so that it is expressed in steroidogenic tissues at the proper time in development, and must be rapidly induced in response to cAMP stimulation. We have summarized the available information concerning the regulation of StAR mRNA levels including promoter mapping and transactivation studies. We also discuss the various transcription factors which have been implicated in the regulation of the StAR gene thus far, and propose models of how StAR transcription may be regulated.

Animals↗
PubMed1999

Steroidogenic acute regulatory (StAR) protein: what's new?

In response to trophic hormone stimulation of steroidogenic adrenal and gonadal cells, the acute biosynthesis of steroid hormones occurs in the order of minutes to tens of minutes and can be contrasted to chronic regulation, which occurs on the order of hours. The steroidogenic acute regulatory (StAR) protein is an indispensable component in the acute regulatory phase and functions by rapidly mediating the transfer of the substrate for all steroid hormones, cholesterol, from the outer to the inner mitochondrial membrane where it is cleaved to pregnenolone, the first steroid formed. This transfer of cholesterol constitutes the rate-limiting step in steroidogenesis. To underscore its importance, mutations in the StAR gene have been shown to be the only cause of the potentially fatal disease lipoid congenital adrenal hyperplasia, in which affected individuals synthesize virtually no steroids. Since the cloning of the murine cDNA in 1994, many observations have substantiated the critical role of StAR in regulated steroidogenesis. The purpose of this review will be to summarize briefly some background material on StAR and then attempt to update several recent and interesting findings on the StAR protein.

Animals↗
PubMed1999

An update on the mechanism of action of the Steroidogenic Acute Regulatory (StAR) protein.

The Steroidogenic Acute Regulatory (StAR) protein has been demonstrated to be an indispensable component in the acute regulation of steroid hormone biosynthesis. The StAR protein, which is rapidly synthesized in response to tropic hormone stimulation, apparently functions by mediating the transfer of the substrate for all steroid hormones, cholesterol, from the outer to the inner mitochondrial membrane where it is cleaved to pregnenolone, the first steroid formed. This transfer is the regulated and rate limiting step in steroidogenesis. Mutations in the StAR gene have been shown to be the only cause of the potentially fatal disease, lipoid congenital adrenal hyperplasia, in which the affected individual can synthesize virtually no steroids. One of the most interesting and important areas of studies on this protein encompasses the mechanism whereby the StAR protein mediates the transfer of cholesterol to the inner mitochondrial membrane. While this mechanism remains unknown at this time, this review will attempt to bring this aspect of StAR function up to date.

Adrenal Hyperplasia, Congenital↗