Lance P. Walsh

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 vitrotranscription/translation was phosphorylated by the protein kinase A catalytic subunit in the presence of [γ-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.

Effects of Lindane on Steroidogenesis and Steroidogenic Acute Regulatory Protein Expression

Abstract Lindane, the γ 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 γ-HCH and its isomers, α-HCH and δ-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 P450scc enzyme. Our studies demonstrate that α-, δ-, and γ-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)2cAMP-stimulated StAR protein levels. Therefore, our results are consistent with the hypothesis that α-, δ-, and γ-HCH inhibited steroidogenesis by reducing StAR protein expression, an action that may contribute to the pathogenesis of lindane-induced reproductive dysfunction.

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.

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.

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.

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.

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.