Oxysterols regulate expression of the steroidogenic acute regulatory protein.

The steroidogenic acute regulatory (StAR) protein promotes intramitochondrial delivery of cholesterol to the cholesterol side-chain cleavage system, which catalyzes the first enzymatic step in all steroid synthesis. Intriguingly, substrate cholesterol derived from lipoprotein can upregulate StAR gene expression. Moreover, substrate oxysterols have been suggested to also play a role. To investigate whether oxysterols can regulate StAR expression, two steroidogenic cell lines, mouse Y1 adrenocortical and MA-10 Leydig tumor cells, were treated with various oxysterols and steroids, including 25-hydroxycholesterol (25 OHC), 22(R)OHC and 20alphaOHC. The majority of these compounds rapidly increased StAR protein levels within as little as 1 h. The most potent oxysterols were 20alphaOHC for Y1 and 25 OHC for MA-10 cells. After 8 h, StAR mRNA abundance also increased whereas there were no detected changes in promoter activity. Thus, in contrast to lipoprotein, oxysterols acutely increase StAR protein levels independently of mRNA abundance, and later increase mRNA levels independently of new gene transcription. Therefore, we propose that oxysterols modulate steroidogenesis at two levels. First, oxysterols may be important in post-transcriptional regulation of StAR activity and production of steroids for paracrine action. Secondly, through direct conversion to steroid, oxysterols may account in part for StAR-independent steroid production in the body.

Protein tyrosine phosphatases are involved in LH/chorionic gonadotropin and 8Br-cAMP regulation of steroidogenesis and StAR protein levels in MA-10 Leydig cells.

The LH signal transduction pathway features the activation of protein tyrosine phosphatases (PTPs) as one of the components of a cascade that includes other well characterized events such as cAMP-dependent protein kinase A (PKA) activation. Moreover, the action of PTPs is required to increase the rate-limiting step in steroid biosynthesis, namely the cAMP-regulated transfer of cholesterol to the inner mitochondrial membrane. Since both PKA activity and steroidogenic acute regulatory (StAR) protein induction are obligatory steps in this transfer of cholesterol, the present study was performed to investigate the role of PTPs in the regulation of PKA activity and StAR expression in response to LH/chorionic gonadotropin (CG) and 8Br-cAMP in MA-10 cells. While the exposure of MA-10 cells to the PTP inhibitor, phenylarsine oxide (PAO), did not modify PKA activity, it partially inhibited the effect of human CG and cAMP analog on StAR protein levels. Time-course studies demonstrated that PAO inhibited cAMP induction of StAR protein and mRNA. At 30 min, the effect on cAMP-stimulated StAR protein levels was a 35% inhibition, progressing to up to 90% inhibition at 120 min of stimulation. The maximal inhibitory effect on cAMP-induced StAR mRNA level was obtained at 60 min (85%). In summary, these results demonstrate that inhibition of PTP activity affected both StAR protein and mRNA synthesis and suggest that the activity of hormone-regulated PTPs is a requirement in the LH signaling cascade that results in the up-regulation of StAR protein and, subsequently, increased steroid synthesis.

Heat shock interferes with steroidogenesis by reducing transcription of the steroidogenic acute regulatory protein gene.

A key regulatory point in fine tuning of steroidogenesis is the synthesis of steroidogenic acute regulatory protein, which transfers cholesterol into mitochondria. Heat shock and toxic insults reduce steroidogenic acute regulatory protein, severely compromising steroid synthesis. As the molecular mechanisms for this reduction remain elusive, we tested the hypothesis that heat shock directly interferes with transcription of the steroidogenic acute regulatory protein gene. We show that, in mouse MA-10 Leydig tumor cells, heat shock caused drastic declines in (Bu)(2)cAMP-induced progesterone accumulation and steroidogenic acute regulatory protein transcript abundance. A proximal steroidogenic acute regulatory protein promoter fragment (-85 to +39) is sufficient to direct both cAMP inducibility and heat shock inhibition. Nuclear extracts from MA-10 cells displayed binding to this proximal promoter fragment as a low mobility complex in gel shift experiments. This complex disappeared in nuclear extracts taken at 5 and 10 min after initiation of heat shock and reappeared in extracts taken at 2 and 8 h. Similar low- mobility complexes formed on oligonucleotides representing the overlapping subfragments of the minimal steroidogenic acute regulatory protein promoter fragment sensitive to the heat shock effect. Extracts from heat-shocked MA-10 cells displayed reduced complex formation to each of the subfragments. We conclude that heat shock reduces progesterone synthesis, steroidogenic acute regulatory protein mRNA abundance, and steroidogenic acute regulatory protein promoter activity and disrupts binding of nuclear proteins to the proximal region of the steroidogenic acute regulatory protein promoter. Together these observations provide strong evidence for a mechanism of transcriptional inhibition in the down-regulation of steroidogenic acute regulatory protein expression by heat shock.

Elements involved in the regulation of the StAR gene.

The steroidogenic acute regulatory protein (StAR) mediates the transfer of cholesterol from the outer to the inner mitochondrial membrane, the regulated step in steroidogenesis. A most interesting facet of this protein is the manner in which its expression is acutely regulated. In this regard, a number of studies have concentrated on the search for consensus cis regulatory elements within its promoter, and, more importantly, on whether these elements are involved in its expression. This short review will summarize some of the findings that have been reported concerning the nature of how the expression of this gene is regulated.

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.

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.

Nigericin inhibits accumulation of the steroidogenic acute regulatory protein but not steroidogenesis.

The steroidogenic acute regulatory (StAR) protein mediates the delivery of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol side chain cleavage complex converts it to pregnenolone. While the mechanism by which this mitochondrial protein acts is poorly understood, one component of the mitochondrial electrochemical gradient, the electrochemical potential (DeltaPsi), appears to be essential. In this study, the importance of the other component, the proton gradient (DeltapH), was examined. Disruption of DeltapH with the electroneutral K(+)/H(+) exchanger, nigericin, had no effect on steroidogenesis in MA-10 mouse Leydig tumor cells at concentrations which significantly reduced StAR protein levels. These data indicate for the first time in true steroidogenic cells, that StAR can act prior to being fully imported into the mitochondria and are consistent with observations made in COS-1 cells using mutant forms of StAR. These results support the hypothesis that a DeltaPsi-dependent factor is required for StAR activity and demonstrate that nigericin is the first compound described, capable of inhibiting StAR accumulation without affecting steroidogenesis.

Expression of adrenocortical steroidogenic acute regulatory (StAR) protein is influenced by chromaffin cells.

Cell-cell interactions are crucial role for the proper functioning of endocrine glands. We recently demonstrated that interactions of chromaffin and cortical cells are important for adrenocortical steroidogenesis. However, the molecular mechanisms have not been elucidated and it is unclear if this involves acute and/or chronic processes. By Northern analysis and the quantitative technique of TaqMan PCR we investigated whether chromaffin cells influence the regulation of StAR and the peripheral benzodiazepine receptor (PBR), both required for the rate-limiting step, the delivery of cholesterol to the inner mitochondrial membrane. StAR mRNA levels in bovine adrenocortical cells were increased by incubation with chromaffin cell-conditioned medium (CCM). Short-term treatment for 4 h resulted in a greater stimulation (229+/-29% of basal, mean+/-SEM) than did longer incubation times of 8 h and 5 days (159+/-13 and 177+/-24%). Neither short nor a long-term treatment affected PBR expression. Consistently, the major secretion of chromaffin cells, epinephrine dose-dependently stimulated StAR expression with no effect on PBR mRNA. In conclusion, adrenomedullary secretory products are not necessary for the maintenance of PBR expression but facilitate steroid biosynthesis by increasing StAR mRNA expression and therefore can account for an ACTH-independent regulation of the rate-limiting step in steroidogenesis.

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.

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.

Econazole and miconazole inhibit steroidogenesis and disrupt steroidogenic acute regulatory (StAR) protein expression post-transcriptionally.

The imidazole antifungal drugs econazole and miconazole have previously been shown to disrupt steroidogenesis in Leydig and adrenal cells by inhibiting 17alpha-hydroxylase/17,20-lyase (P450c17) enzyme activity, thus reducing the conversion of progesterone to androstenedione. However, a recent study in Y-1 adrenal cells indicated that these compounds may also reduce the availability of cholesterol to the cytochrome P450 side chain cleavage (P450(scc)) enzyme, the first enzyme in the steroidogenic pathway. Since the steroidogenic acute regulatory protein (StAR) mediates the transfer of cholesterol from the outer to the inner mitochondrial membrane where the P450(scc) enzyme resides, an action which constitutes the rate-limiting and acutely-regulated step in steroidogenesis, we hypothesized that these drugs may also reduce StAR expression and/or activity. Our studies demonstrate that these drugs reversibly inhibited (Bu)(2)cAMP-stimulated progesterone production in a dose- and time-dependent manner in MA-10 cells without affecting total protein synthesis or P450(scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzyme expression or activity. In contrast, they dramatically decreased (Bu)(2)cAMP-stimulated StAR protein expression post-transcriptionally. This study indicates that StAR protein is susceptible to inhibition by at least some imidazole compounds that inhibit steroidogenesis.

The role of arachidonic acid on LH-stimulated steroidogenesis and steroidogenic acute regulatory protein accumulation in MA-10 mouse Leydig tumor cells.

Metabolic pathways leading to the production of arachidonic acid (AA) and its metabolites have been reported to have modulatory effects on steroidogenesis in a number of cell types. To examine the importance of the arachidonic acid pathway in steroid production and steroidogenic acute regulatory (StAR) protein expression, luteinizing hormones (LH) or N6-2-o-dibutyryl-adenosine-3:5-cyclic monophosphate-(Bt2cAMP) stimulated MA-10 mouse Leydig tumor cells were treated with various concentrations of quinacrine (an inhibitor of arachidonic acid production). Incubation of the cells with quinacrine resulted in dose-dependent decreases in steroid production and StAR protein. Twenty micromolars quinacrine inhibited 92 and 91% of LH-induced progesterone and StAR protein, respectively, and 98 and 90% of Bt2cAMP-induced progesterone and StAR protein. Reversal of this inhibition was obtained by incubation of quinacrine-treated cells with various levels of AA, which resulted in a dose-dependent increase in both steroid and StAR protein levels. Two hundred micromolars of AA rescued 57 and 60% of the LH-induced steroid production and StAR protein, respectively, and 52 and 89% of Bt2cAMP-induced steroid production and StAR protein. These results suggest that the effect of AA on LH- and cAMP-stimulated steroidogenesis is associated with the modulation of StAR protein expression.

Induction of differentiation and apoptosis by sodium selenite in human colonic carcinoma cells (HT29).

To explore the mechanism(s) by which selenium (Se) exerts its cancer chemopreventive activity, we studied the effect of selenite (0-100 microM) on cell growth, viability, differentiation, detachment, DNA fragmentation and apoptosis in human colonic carcinoma cells (HT29). Selenite (>5 microM) decreased cell growth, increased cell detachment and decreased intracellular levels of reduced glutathione (GSH), whereas >10 microM selenite induced cell differentiation and apoptosis. The chemopreventive effects of selenite may be related in part to the generation of reactive oxygen species (ROS) resulting from the reaction between selenite and GSH.

Phosphorylation of steroidogenic acute regulatory protein (StAR) modulates its steroidogenic activity.

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone synthesis. StAR is thought to increase the delivery of cholesterol to the inner mitochondrial membrane where P450scc resides. Tropic hormones acting through the intermediacy of cAMP rapidly increase pregnenolone synthesis, and this rapid steroidogenic response is believed to be due to StAR's action. The StAR protein contains two consensus sequences for phosphorylation catalyzed by protein kinase A that are conserved across all species in which the amino acid sequence of the StAR protein has been determined. We demonstrated that human StAR expressed in COS-1 cells exists in at least four species detectable by two-dimensional gel electrophoresis followed by Western blotting. The two more acidic species disappeared after treatment of the cell extracts with alkaline phosphatase. 32P was incorporated into StAR protein immunoprecipitated from COS-1 cell extracts, and a 10-min treatment with 8-bromo-cAMP increased 32P incorporation into the StAR preprotein. StAR protein generated by in vitro transcription/translation was phosphorylated by the protein kinase A catalytic subunit in the presence of [gamma-32P]ATP. Mutation of potential sites for protein kinase A-mediated phosphorylation at serine 57 and serine 195 to alanines, individually, reduced 32P incorporation from labeled ATP into StAR preprotein produced by in vitro transcription/translation when incubated with protein kinase A catalytic subunit. 32P labeling of StAR protein expressed in COS-1 cells was also reduced when serine 57 or serine 195 were mutated to alanines. A double mutant in which both serine 57 and serine 195 were changed to alanines displayed markedly reduced 32P incorporation. To determine the functional significance of StAR phosphorylation, we tested the steroidogenic activity of the wild-type StAR and mutated StAR proteins in COS-1 cells expressing the human cholesterol side chain cleavage enzyme system. Mutation of the conserved protein kinase A phosphorylation site at serine 57 had no effect on pregnenolone synthesis. However, mutation of the serine residue at 195 resulted in an approximately 50% reduction in pregnenolone production. The S195A mutant construct did not yield the more acidic species of StAR detected in two-dimensional Western blots, indicating that the mutation affected the ability of the protein to be post-translationally modified. Mutation of the corresponding serine residues in murine StAR (Ser56 and Ser194) to alanines yielded results that were similar to those obtained with human StAR; the S56A mutant displayed a modest reduction in steroidogenic activity, whereas the S194A mutant had approximately 40% of the activity of murine wild-type StAR. In contrast to the human S195A mutation, conversion of serine 195 to an aspartic acid residue had no effect on steroidogenic activity, consistent with the idea that a negative charge at this site modulates StAR function. Our observations suggest that phosphorylation of serine 194/195 increases the biological activity of StAR and that this post- or co-translational event accounts, in part, for the immediate effects of cAMP on steroid production.

Lymphokine-activated killer (LAK) cells modulate the effects of IL-2 on a T cell-mediated immune response.

The ability of LAK cells and/or IL-2 to affect the course of an established T cell response was examined in a delayed-type hypersensitivity (DTH) model. IL-2 greatly increased the magnitude of the response at 24 h, while LAK cells alone had no effect. The administration of LAK cells and IL-2 together also had no effect on the magnitude of the DTH response, demonstrating that LAK cells were able to remove the enhancement seen with IL-2 alone. The presence of LAK cells reduced the serum half-life of IL-2 significantly, but not to an extent able to account for the observed loss of IL-2 induced DTH enhancement. IL-2 administration influenced cell phenotypes in the spleen and draining lymph nodes (DLN), as well as increasing splenic weight; the additional presence of LAK cells markedly altered these effects of IL-2 in the spleen (but not the DLN). Taken together, these results suggest that LAK cells interact with activated T-cells within the immune system and modulate their function.

Multiple epitopes of the human ovarian cancer antigen 14C1 recognised by human IgG antibodies: their potential in immunotherapy.

We have defined a novel ovarian cancer-associated membrane antigen, 14C1, using human monoclonal antibodies derived by EBV-transformation of in situ sensitised patients' B-cells. The pattern of recognition of this antigen by these antibodies suggests that at least three epitopes are discernable. These antibodies can be used to promote the in vitro killing of ovarian cancer cells by activated macrophages and cytokines, implying a role for this antigen in the immunotherapy of ovarian malignancies. Evidence is presented that the 14C1 antigen may have some transmembrane signalling function.

Enhancement of a delayed hypersensitivity reaction to a contact allergen, by the systemic administration of interleukin-2.

The immunopharmacological effects of interleukin-2 (IL-2) on the sensitization and effector phases of the delayed-type hypersensitivity (DTH) reaction were studied using contact sensitivity to the haptenizing agent dinitrochlorobenzene (DNCB). When administered at the time of priming to DNCB, IL-2 had no effect on the subsequent magnitude of the response. Interleukin-2 was, however, able to increase the magnitude of the response when given at the time of secondary challenge; the degree of change was directly related to the dose of IL-2. The proportions of T cells in the draining lymph node and spleen of IL-2-treated animals decreased by approximately one-third, but there was no alteration to the balance between CD4+ and CD8+ T cells. The results suggest that the increase in DTH observed was due to a pharmacological effect rather than to an increase in T-cell number.

14C1, an antigen associated with human ovarian cancer, defined using a human IgG monoclonal antibody.

We have selected a human EBV-transformed cell line from the involved lymph nodes of an ovarian cancer patient which secretes an IgG1 kappa antibody, able to recognize an antigen present on the surface of ovarian cancer cells. The antigen, termed '14Cl,' has previously been shown by immunohistological techniques to be present on the surface of the malignant cells within tumour specimens. Western blotting analysis has shown that the majority of primary ovarian cancer specimens and three continuous cell lines derived therefrom express 14Cl; other tissue types were negative. Preliminary biochemical characterization has been carried out, which shows that the 14Cl antigen has a molecular weight range of 25-32 kD and an isoelectric point from pI 6.3 to 6.8. We believe that the 14Cl antigen is immunologically relevant to ovarian cancer patients and may therefore represent a novel target for both active and passive immunotherapy.