Insights into steroidogenic acute regulatory protein (StAR)-dependent cholesterol transfer in mitochondria: evidence from molecular modeling and structure-based thermodynamics supporting the existence of partially unfolded states of StAR.

The steroidogenic acute regulatory protein (StAR) is the major entrance for cholesterol in mitochondria under acute stimulation. Under such circumstances, dysfunctional StAR activity can ultimately lead to lipoid congenital adrenal hyperplasia (LCAH). A complete understanding of the StAR's molecular structure and mechanism is essential to comprehend LCAH. Thus far, there is no mechanistic model that can explain experimental results at the molecular level. This is partly due to the lack of the molecular structure of StAR. The closest approximation to the StAR molecular structure is the human MLN64 which has a similar activity to StAR, has a highly homologous primary structure and for which an X-ray structure is known. In this context, we have modeled the structure of StAR through standard homology modeling procedures based on the MLN64 structure. Our StAR model shows the presence of a hydrophobic cavity of 783.9 A(2) in surface area, large enough to fit one molecule of cholesterol. In addition, we have identified a unique charged pair, as in MLN64, lining the surface of the cavity and which could play a key role in the binding of cholesterol through the formation of an H-bond with its OH moiety. This suggests that the cholesterol-binding site of StAR is located inside this cavity. Taking into account that internal cavities are destabilizing to native protein structures and that the lining of the cavity has to become accessible in order to allow cholesterol binding, we have explored the possibility that StAR could exist in equilibrium with partially unfolded states. Using a structure-based thermodynamics approach, we show that partially folded states (with an unfolded C-terminal alpha-helix, and an open cavity) can be significantly populated at equilibrium and therefore allow cholesterol binding. These results are supported by recent experiments that show a loss of StAR helical character upon binding of an analog of cholesterol. Moreover, we show that the replacement of the residues involved in the charged-pair located in the binding site results in the loss of StAR activity, supporting a key role for these residues. Taken together, our results are applicable to StAR functioning both in the mitochondrial intermembrane space as well as outside the mitochondria.

Fibronectin, laminin, and collagen IV interact with ACTH and angiotensin II to dictate specific cell behavior and secretion in human fetal adrenal cells in culture.

Whereas collagen IV is expressed throughout the fetal adrenal gland during the second trimester of human development, fibronectin, and laminin demonstrate a rather mirror-image distribution, with higher expression of fibronectin in the central portion and laminin at the periphery of the gland. In the present study, extracellular matrices were able to modulate the profile of steroid secretion in primary cultures: collagen IV favored cortisol secretion following adrenocorticotropin (ACTH) or angiotensin II (Ang II) stimulation while specific stimulation of the AT2 receptor of Ang II elicited dehydroepiandrostenedione (DHEA) production. These effects were correlated by changes in mRNA levels of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and cytochrome P450C17. In contrast, fibronectin and laminin decreased cell responsiveness to ACTH in terms of cortisol secretion, but enhanced ACTH-stimulated androgen secretion. Finally, extracellular matrices were able to orchestrate cell behavior: collagen IV and laminin enhanced cell proliferation whereas fibronectin incited cell death. These results indicate that the nature of extracellular matrix coordinates specific steroidogenic pathways and cell turnover in the developing human fetal adrenal gland.

Identification of extracellular matrix components and their integrin receptors in the human fetal adrenal gland.

The development of the human fetal adrenal gland is characterized by a gradient of mitotic activity, cell migration, and cell apoptosis, all of which dictate its particular function. Such plasticity may possibly be under the control of the extracellular environment. The goal of this study was to identify components of the extracellular matrix in second-trimester fetal adrenal glands. Whereas collagen IV was expressed evenly throughout the gland, both fibronectin and laminin demonstrated a mirror-imaged distribution, with higher expression of fibronectin in the central portion and laminin at the periphery of the gland. The integrin subunit alpha1 was found mainly in the definitive zone and the alpha2-subunit mainly in the transitional zone, whereas integrin alpha3 (which binds both fibronectin and laminin) was detected only in the fetal zone. The beta2-subunit was observed solely in chromaffin cells. Such specific gradients of integrin and MEC component expression suggest that the extracellular environment does play a definite role during adrenal gland development. Indeed, compared with that in untreated plastic dishes, ACTH stimulation of dehydroepiandrosterone sulfate and cortisol was enhanced by collagen IV. In addition, fibronectin enhanced dehydroepiandrosterone sulfate but decreased cortisol secretion, compared with collagen IV substrates. These results provide fundamental insight into the contribution of the microenvironment in cellular processes leading to fetal adrenal gland development.

Control of CYP11B2 gene expression through differential regulation of its promoter by atypical and conventional protein kinase C isoforms.

We reported previously that the protein kinase C (PKC) inhibitor GF109203X stimulated the hamster CYP11B2 promoter activity in transfected NCI-H295 cells. PKCalpha, -epsilon, and -zeta were detected in hamster adrenal zona glomerulosa and NCI-H295 cells, and PKCtheta in NCI-H295 cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) inhibited basal and stimulated cytochrome P450 aldosterone synthase mRNA expression by angiotensin (AII), dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP), or KCl in NCI-H295 cells. Basal CYP11B2 promoter activity was inhibited in cells cotransfected with constitutively active (CA) PKCalpha, -epsilon, and -theta mutants, whereas it was increased with CA-PKCzeta. Dominant negative (DN) PKCalpha, -theta, -epsilon, and -zeta mutants stimulated the promoter activity. AII-, KCl-, and Bt2cAMP-stimulatory effects were abolished in cells cotransfected with CA-PKCalpha, -epsilon, or -theta. The effect of Bt2cAMP was abolished by CA-PKCzeta but AII and KCl were still able to enhance the promoter activity. DN-PKCalpha, -epsilon, -theta, or -zeta did not inhibit these effects. Gö6976 enhanced promoter activity, providing further evidence that PKCalpha was involved. Various CYP11B2 promoter constructs were used to identify the area associated with TPA and PKC inhibition. TPA and CA-PKCalpha, -epsilon, or -theta abolished the effects of AII, KCl, and Bt2cAMP on the activity of -102 and longer constructs. In summary, our findings suggest that the hamster CYP11B2 gene is under differential control by conventional (alpha) and atypical (zeta) PKC.

Localization of G protein alpha-subunits in the human fetal adrenal gland.

The aim of the present study was to investigate the presence and localization of the main G protein alpha-subunits in the human fetal adrenal gland during the second trimester of gestation. Immunofluorescence studies conducted on sections from frozen glands obtained immediately after therapeutic abortion indicated that the alpha s subunit of the heterotrimeric Gs protein was detected in all adrenal cell types, except for endothelial cells. The other alpha-subunits had a more specific pattern of distribution. Indeed, the alpha il-2 protein was restricted to the definitive zone, whereas alpha i3 labeling was mainly expressed in the fetal zone. The alpha q protein subunit was localized in vascular endothelial cells at the periphery of the adrenal gland and in fetal cells at the center. Finally, chromaffin cells expressed alpha s, alpha q, and alpha o1, but not alpha o2 nor alpha i. Altogether, these results indicate that the human fetal adrenal gland is not only unique in its particular morphology and expression of steroidogenic enzymes, but also by the differential expression of G protein alpha-subunits. Such cell specific distribution in glands from midgestational fetuses may account for the absence or the different responses to stimuli, when compared with the adult adrenal gland.

Age-related decrease of dehydroepiandrosterone concentrations in low density lipoproteins and its role in the susceptibility of low density lipoproteins to lipid peroxidation.

The incidence of atherosclerosis and related diseases increases with age. The aging process may enhance lipoprotein modification, which leads to an increase in the susceptibility of low density lipoprotein (LDL) and high density lipoprotein (HDL) to oxidation. Dehydroepiandrosterone (DHEA), the most abundant steroid hormone in humans, has been shown to have antiatherogenic effects. This hormone also decreases dramatically with age. In the present study, we were interested in determining the presence of DHEA/DHEAS (dehydroepiandrosterone sulfate) and changes in their concentrations in HDL and LDL lipoproteins with age. Moreover, we studied the susceptibility of LDL to oxidation with age in the presence or absence of vitamin E or DHEA. We demonstrated that vitamin E is unable to restore the decreased resistance to oxidation of LDL from elderly subjects to that of LDL obtained from young subjects. Furthermore, our results provide evidence that DHEA is an integral part of LDL and HDL and disappears to almost nondetectable levels during aging. The DHEA incorporated into the LDL from elderly subjects increased LDL resistance to oxidation in a concentration-dependent manner. The increased resistance provided by DHEA was higher than that with vitamin E. DHEA seems to act either by protecting vitamin E from disappearance from LDL under oxidation or by scavenging directly the free radicals produced during the oxidative process. Our results suggests that DHEA exerts an antioxidative effect on LDL, which could have antiatherogenic consequences. Careful clinical trials of DHEA replacement should determine whether this ex vivo effect could be translated into any measurable antiatherogenic (cardioprotective) action.

Occurrence and effect of PACAP in the human fetal adrenal gland.

In the study reported in this paper, we characterized PACAP in the human fetal adrenal gland and we investigated the effect of PACAP on steroid secretion from cultured fetal adrenal cells. The adrenal gland from 20-week-old fetuses contained substantial concentrations of PACAP-immunoreactive material (88.6 ng/g wet tissue). HPLC analysis of adrenal extracts revealed the presence of both PACAP27 and PACAP38, the latter being the predominant form. Incubation of cultured fetal adrenal cells with PACAP38 (10(-7) M) significantly increased cortisol and DHEAS secretion. Administration of the beta-adrenoreceptor agonist isoproterenol mimicked the stimulatory effect of PACAP on both steroid secretion whereas preincubation of fetal cells with the beta-adrenoreceptor antagonist propranolol suppressed the steroidogenic effect of PACAP. These data, together with the observation that PACAP receptors are exclusively located on chromaffin cells, suggest that, in the fetal human adrenal gland, the effect of PACAP on steroid secretion is mediated via the local release of catecholamines.

Regulation of CYP11B2 gene expression by protein kinase C.

Bisindolylmaleimide, a protein kinase C (PKC) inhibitor, was shown to stimulate the hamster CYP11B2 promoter activity in transfected NCI-H295 cells. In this study we have found that TPA, an activator of PKC, also inhibited the hamster CYP11B2 promoter activity. DAG-dependent PKC alpha and PKC epsilon, and atypical PKC zeta were detected in hamster adrenal zona glomerulosa, whereas the isoforms alpha, epsilon, zeta and theta were found in NCI-H295 cells. CYP11B2 promoter activity was inhibited in cells co-transfected with constitutively active PKC alpha and epsilon mutants, whereas it was increased with the constitutively active PKC zeta mutant. Dominant negative PKC alpha, epsilon and zeta mutants stimulated the promoter activity. Gö6976, a specific inhibitor of classical PKCs, enhanced promoter activity, providing further evidence that PKC alpha, the only classical PKC revealed in hamster adrenal and NCI-H295 cells, was involved in the promoter inhibition.

Molecular modeling of the hamster adrenal P450C17.

The cytochrome P450C17 (C17) is the steroidogenic enzyme responsible for the conversion of pregnenolone and progesterone to dehydroepiandrosterone (DHEA) and delta4-androstenedione (AD) respectively. This conversion is achieved by two enzymatic activities, 17alpha-hydroxylase and 17,20-lyase, located at the same active site. In man, the adrenal C17 basically only produces DHEA. We have shown that the hamster adrenal C17 produces DHEA as well as AD. Moreover, the hamster like man produces cortisol as its major glucocorticoid. We can thus compare the hamster and human adrenal C17, and use their differences in order to elaborate a strategy for structure-function studies. We have thus engineered hamster adrenal C17 mutants which possess modified enzymatic activities. We also proceeded to elaborate a three-dimensional model of the hamster C17 to visualise the structural impact of these mutations. This model demonstrates that the mutations created are not localised at the active site, but rather in surrounding regions. These could affect the conformation of the active site, in turn, modulating the 17alpha-hydroxylase and 17,20-lyase activities. For example, the mutation T202N is located next to Val 482 and Val 483 which compose the roof of the active site. This mutation decreased both 17alpha-hydroxylase and 17,20-lyase activities, indicating the importance of the roof of the active site for general functionality of the C17.

Involvement of the angiotensin II type 2 receptor in apoptosis during human fetal adrenal gland development.

The aim of this study was to establish a link between the highly expressed angiotensin II (Ang II) type 2 receptor (AT2) in human fetal adrenal cells and the proposed apoptotic activity in the center of the gland. There was an important increase in apoptotic DNA fragmentation with age in adrenal glands of fetuses from 15-20 weeks gestation. Adrenal cells showing the characteristic apoptotic internucleosomal DNA fragmentation were localized in the central portion of the fetal zone. In cells cultured for 24 h, Ang II, via the AT2 receptor, induced DNA fragmentation and cleavage of the DNA repair enzyme, poly-(ADP-ribose) polymerase. Furthermore, characteristic membrane blebbing was observed specifically on cells of the fetal zone. Immunofluorescence studies demonstrated that stimulation with Ang II or CGP 42112 (an agonist of the AT2 receptor) strongly modified the actin network, now localized exclusively along the plasma membrane, with a predominance of labeling at the base of the bleb formation. This rearrangement of actin distribution was different in cells from the definitive zone, corroborating the observation that these cells express many more Ang II type 1 receptors (AT1) than AT2 receptors. Taken together, our data indicate that the AT2 receptor is involved in the apoptotic process observed in the human fetal adrenal gland and could participate in the morphological changes occurring after birth, leading to involution of the fetal zone.

The in vivo effects of adrenocorticotropin and sodium restriction on the formation of the different species of steroidogenic acute regulatory protein in rat adrenal.

We have studied the in vivo expression of steroidogenic acute regulatory protein (StAR) in adrenals of control, ACTH-treated, and Na+-restricted rats. Indirect immunofluorescence by microscopy revealed the presence of StAR in the zonae glomerulosa (ZG) and fasciculata-reticularis (ZFR). An increased signal was observed in the ZG and zona fasciculata, 5 h after ACTH injection; a few cells of the medulla were also positive. Immunogold electron microscopy showed that StAR was mainly located over mitochondria (MT). By immunoblotting, a major 29-kDa and other minor StAR bands migrating between 30 and 39 kDa were increased 5 h after ACTH treatment but remained unchanged after 1 h. By two-dimensional-PAGE, four StAR species were revealed in homogenates of control ZG, and their intensity was increased 5 h after ACTH treatment but not after 1 h. Also, additional acidic species were seen 5 h after treatment. Other bands with basic isoelectric point were revealed between 29 and 37 kDa. Analyses on whole gland MT and supernatant (SN) revealed four bands in the control SN and five in ACTH SN; the intensity of one band was increased, and that of another one was decreased, in SN of treated rats. ACTH treatment resulted in the localization of many low-isoelectric point StARs in MT. After two-dimensional-PAGE, differences were found in the mobility of some StAR species in the ZG between controls and Na+-restricted rats. In MT, four bands were revealed in the ZG preparations of Na+-restricted and two bands in controls. Four bands were revealed in the ZG SNs of control and Na+-restricted rats; an additional band was observed only in the SN of treated animals, whereas the intensity of another band decreased. Na+ restriction did not affect StAR in the ZFR. In conclusion, StAR was present in the rat adrenal cortex ZG and ZFR and was mainly located in MT. StAR expression was inducible in the ZG and the ZF by ACTH, resulting in the formation of many StAR acidic species; interestingly, such changes were detectable 5 h, but not 1 h, after ACTH administration, suggesting that steroidogenesis stimulation by StAR might occur mainly outside MT. Although less spectacular than for ACTH, Na+ restriction also affected StAR expression in the ZG but not in the ZFR, by increasing two mitochondrial and one SN species, implying that StAR is involved in the mechanism of action of Na+ restriction in promoting aldosterone formation. These results suggest that differential processing and/or changes in phosphorylation may occur in vivo upon ACTH treatment and Na+ restriction. We hypothesize that modification of a relatively small quantity of StAR, mainly located outside MT, is necessary to increase adrenal steroidogenesis challenged either by ACTH or Na+ restriction.

Mutations in the gene encoding the alpha-subunit of the Gs protein in molar pregnancy.

Molar pregnancy is a gestational trophoblastic disease associated with a trophoblastic proliferation and a protein synthesis alteration. It is characterized by the presence of hydatiform moles, which are fluid-filled cysts derived from the chorionic villi of the placenta. Recent studies have reported a reduced expression of several types of G proteins including Gsalpha in molar pregnancies suggesting alterations in G protein structure in hydatiform moles. To identify mutations that lead to Gsalpha deficiency, we isolated genomic DNA from hydatiform moles and used polymerase chain reaction to amplify all exons of the Gsalpha gene. Amplified Gsalpha gene fragments were analyzed by sequencing using the dideoxy chain termination method. Tissues obtained from three complete hydatiform moles and one partial hydatiform mole were examined. We have identified a heterozygous 8-bp deletion in exon 10 of the Gsalpha gene, in two complete hydatiform moles, that had evidence for a dysfunctional Gsalpha protein. This deletion produced a truncated protein. We have also identified a heterozygous polymorphism in exon 5 in two complete hydatiform moles, and a homozygous substitution (A-->G) in intron 5 of the Gsalpha gene in the other complete hydatiform mole; these two last types of mutations should not have any effects on protein activity.

Inhibitory effects of Serenoa repens on the kinetic of pig prostatic microsomal 5alpha-reductase activity.

The pathogenesis of benign prostatic hyperplasia is linked to the accumulation of dihydrotestosterone (DHT), the active form of testosterone (T), in prostatic tissue. We have defined characteristics of 5alpha-reductase enzyme which catalyzes the conversion of T into DHT in prostatic microsomes of growing pigs. Peaks for the 5alpha-reductase activity were found at pH 5.5 and 8.0, which indicates the presence of both type 1 and type 2 isozymes. Kinetic parameters of porcine 5alpha-reductase in the presence of Serenoa repens extracts revealed uncompetitive, noncompetitive, and mixed types of inhibitions. Our results show the inhibitory action of S. repens on prostate porcine microsomal 5alpha-reductase activity.

In vivo effects of adrenocorticotrophin on the expression of the hamster steroidogenic acute regulatory protein.

In this study, we report the cDNA cloning of hamster adrenal steroidogenic acute regulatory (StAR) protein and the effect of adrenocorticotrophin (ACTH) on its expression in vivo. A hamster adrenal cDNA library was screened using an 852 bp fragment obtained by polymerase chain reaction; this fragment corresponds to the entire coding sequence (CDS) of the hamster adrenal StAR cDNA. Ten clones of different lengths were isolated and sequenced. The longest clone was 1564 bp and contained 34 bp in the 5'-untranslated region, 852 bp in the CDS, and 678 bp in the 3'-untranslated region (3'-UTR). Two polyadenylation signal sequences were found in the 3'-UTR. The CDS of the ten isolated clones was identical, but six of these lacked the last 132 nucleotides in the 3'-UTR, thus indicating that they had used the first polyadenylation signal. The hamster StAR protein contains 284 amino acid residues, and is 91.9% homologous to mouse, 90.5% to rat, 86.4% to human, 85% to porcine, and 82.5% to bovine StAR protein. Southern blot analysis indicated the presence of only one StAR gene in the hamster genome. Northern blotting analysis revealed the presence of the StAR mRNA in male and female steroidogenic tissues, namely adrenals and gonads, but not in the liver or in the kidneys of either sex. Three mRNA species of 1.7, 3.1 and 5.3 kb were found in whole hamster adrenals. Administration of ACTH to hamsters provoked increases (two- to threefold) in the adrenal content of the StAR mRNA within 1 h in vivo. Western blotting analysis on adrenal mitochondria showed that the level of StAR protein was also significantly elevated (1.5-fold) 1 h after ACTH treatment.

The acute and chronic effects of adrenocorticotropin on the levels of messenger ribonucleic acid and protein of steroidogenic enzymes in rat adrenal in vivo.

The purpose of this study was to evaluate the effects of acute (a single injection) and chronic stimulation (twice daily injection for 9 days) by ACTH on changes occurring in the temporal expression of steroidogenic enzymes in the rat adrenal in vivo. Under acute ACTH stimulation, the level of steroidogenic acute regulatory protein (StAR) messenger RNA (mRNA) was increased within 0.5 h in both zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR), with maximal increases of 220-370% and 300-350% in the ZG and ZFR, respectively. Increases in the levels of StAR protein in homogenates were also found in the ZG (700%) and the ZFR (300%), but were delayed compared with those of their mRNA. Furthermore, the increase in mitochondrial StAR protein was concomitant with that in the homogenate, indicating that the entry of StAR into mitochondria might not be necessary to increase steroidogenesis during the early stimulatory phase. The levels of c-jun, c-fos, junB, and fosB mRNA in ZG and ZFR were also rapidly maximally elevated within 0.5-1 h after ACTH administration and fell to near control levels 5 h posttreatment. The levels of c-jun protein were already increased in both zones at 1 h, reached 200% at 3 h, and remained elevated 5 h post-ACTH treatment. The levels of c-Fos protein were maximally increased by 240% in both zones after 1 h and decreased thereafter to control values at 5 h. Few changes were observed in the adrenal protein contents of cholesterol side-chain cleavage cytochrome P450 (P450scc), cytochrome P450 11beta-hydroxylase (P450C11), cytochrome P450 21-hydroxylase (P450C21), and 3beta-hydroxysteroid dehydrogenase (3betaHSD). Under chronic stimulation by ACTH, we observed elevations in the levels of plasma corticosteroids and changes in the mRNA and protein levels of many adrenal steroidogenic enzymes in both zones. In the ZG, administration of ACTH for 9 days provoked an increase in the level of StAR mRNA (210-270%) and a decrease in the levels of 3betaHSD, cytochrome P450 aldosterone synthase (P450aldo), and AT1 receptor mRNA (by 40%, 70%, and 90%, respectively), whereas the levels of P450scc and P450C21 mRNA did not differ significantly from the control values. Western blotting analysis showed that the adrenal ZG protein levels of StAR and P450scc were increased (150%), 3betaHSD was not changed, and P450C21 was decreased by 70%. In the ZFR, the levels of P450scc and StAR mRNAs were increased (260% and 570-870%, respectively). The levels of 3betaHSD, P450C21, and P450C11 mRNA did not differ from control values in that zone. Western blotting analysis showed that the ZFR protein level of 3betaHSD was not changed, P450scc and P450C21 were decreased by 40% and 60%, respectively, and StAR was increased by 160%. Although c-fos and fosB mRNAs were undetectable after 9 days of chronic ACTH treatment, c-jun mRNA and its protein were still detectable, suggesting a basic role for this protooncogene in maintaining the integrity and function of the adrenal cortex. When dexamethasone was administered to rats for 5 days to inhibit their ACTH secretion, the mRNA levels of many steroidogenic enzymes were decreased, with the exception of StAR, 3betaHSD, and P450aldo. These results confirm the importance of physiological concentrations of ACTH in maintaining normal levels of adrenocortical enzymes and also indicate that in addition to ACTH, other factors are involved in controlling the expression of StAR, 3betaHSD, and P450aldo. In conclusion, we showed that ACTH acutely increases StAR mRNA followed, after a delay, by an increase in the level of StAR protein; this suggests that posttranslational modifications of the StAR precursor occurred during the early stimulatory phase and before the apparent translation of the newly formed mRNA. The rapid induction of protooncogenes suggests their participation in the action of ACTH to stimulate steroidogenesis. (ABSTRACT TRUNCATED)

Transcriptional activity of the hamster CYP11B2 promoter in NCI-H295 cells stimulated by angiotensin II, potassium, forskolin and bisindolylmaleimide.

We studied the regulation of the hamster CYP11B2 gene in the NCI-H295 cell line, which is known to produce aldosterone in response to stimulation by angiotensin II (AII) and KCl. Ten deletion plasmids harboring the 5'-untranslated region of the CYP11B2 gene were used for chloramphenicol acetyltransferase (CAT) assays. Transient transfections showed progressively increasing basal promoter activity by constructs beyond the TATA box, with a peak occurring with the -167 bp construct which contains putative Adl, Ad2, Ad5 and the newly reported -143/-161 cis-element sequences. The promoter activity was lower with the construct containing the putative Ad3 cis-element and increased with longer constructs. This indicates the presence of both inhibitory and stimulatory cis-elements in this area of the gene. Expression of the reporter gene of all constructs was stimulated by AII and KCl, with the exception of the construct containing only the TATA box, which showed 6-fold and 10-fold increases occurring with the -167 bp deletion plasmid. The patterns of increase in CAT activity with AII and KCI treatment were similar, showing that these two regulators can stimulate hamster CYP11B2 promoter activity through common cis-elements. The calcium channel antagonist nifedipine blocked the stimulatory effects of KCl on CAT activity, showing the involvement of calcium channels in the regulation of CYP11B2 gene transcription by KCl. 12-O-Tetradecanoylphorbol 13-acetate, a known stimulator of the protein kinase C (PKC) signaling pathway, was without significant effect on CAT activity. Bisindolylmaleimide, a specific inhibitor of PKC, had a significant enhancing effect (3.4- to 6-fold), indicating that PKC may negatively regulate the expression of the hamster CYP11B2 gene in NCI-H295 cells. A mutation was induced in the sequence -143/-161 of the - 350 bp construct in order to determine its importance in the regulation of hamster CYP11B2 promoter activity. The stimulatory effects of AII, KCl, forskolin and bisindolylmaleimide on CAT activity were significantly less in the mutant than in the wild type. These results confirm that this cis-element is necessary in maintaining a high level of transcriptional activity in stimulated NCI-295H cells. In conclusion, using NCI-295H transfected cells, we have found that the 5'-untranslated region of the hamster CYP11B2 gene possesses transcriptional activity with stimulatory and also inhibitory cis-elements; CYP11B2 promoter activity can be stimulated by AII, KCl, forskolin, dibutyryl cAMP and bisindolylmaleimide. Our results suggest that this gene is positively regulated through the protein kinase A signaling pathway and through calcium channels, whereas PKC may have a negative regulatory effect upon the transcription of the CYP11B2 gene. Furthermore, we have shown that the cis-element -143/-161 in the 5'-untranslated region of the hamster CYP11B2 gene is important in maintaining a high level of promoter activity in stimulated NCI-295H cells.

Localization, characterization, and second messenger coupling of pituitary adenylate cyclase-activating polypeptide receptors in the fetal human adrenal gland during the second trimester of gestation.

The distribution and pharmacological properties of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors were studied in the fetal human adrenal gland during the second trimester of gestation. Autoradiographic studies, using [125I]PACAP27 as a radioligand, revealed that PACAP-binding sites are exclusively located on chromaffin cells of adrenals from fetuses 14-20 weeks old. Biochemical characterization of binding revealed the occurrence of a single class of PACAP-binding sites with a dissociation constant value of 0.32-0.74 nmol/L and a binding capacity of 0.30-0.81 pmol/mg wet tissue. PACAP27 and PACAP38 were equipotent in competing for [125I]PACAP27 binding (IC50 = 0.28-0.64 nmol/L and 0.15-0.81 nmol/L, respectively), and the Hill coefficients were close to 1. In contrast, vasoactive intestinal polypeptide was much less efficient in displacing the tracer (IC50 = 4-362 nmol/L), and the Hill coefficients were less than 0.6. PACAP38 induced a dose-dependent increase in cAMP production in fetal human adrenal cell suspension (ED50 = 0.07 +/- 0.02 nmol/L), as well as in cells maintained in culture for 5 days (5.4 +/- 1.8 nmol/L). In contrast, PACAP38 induced a modest increase in inositol phosphate formation. These data indicate that type I PACAP receptors are present in the early stages of the human medulla organization during the process of migration of chromaffin cells from the periphery to the central part of the gland. The present results suggest that PACAP could be involved in the regulation of the human adrenochromaffin cells during ontogenesis.

Dehydroepiandrosterone protects low density lipoproteins against peroxidation by free radicals produced by gamma-radiolysis of ethanol-water mixtures.

Oxidized low density lipoproteins (LDL) are believed to play a central role in the events that initiate atherosclerosis. Antioxidants have been shown to decrease the oxidation of LDL, leading to the diminution of atherosclerosis. Since it is well-known that decreased levels of dehydroepiandrosterone (DHEA) are linked to the development of atherosclerosis, we studied the modulation of the oxidation of LDL by DHEA. LDL were obtained from 10 healthy subjects and oxidized by free radicals produced by gamma-radiolysis of ethanol-water mixtures. The formation of conjugated dienes and thiobarbituric acid-reactive substances (TBARS), the vitamin E content, as well as the incorporation of 4-[14C]DHEA in LDL and the chemotactic effect of oxidized LDL in the presence of DHEA towards monocytes, were investigated. It was found that DHEA was able to inhibit the oxidation of LDL by reducing over 90% of the conjugated dienes and TBARS formation, as well as by reducing the vitamin E disappearance and significantly decreasing the chemotactic activity towards monocytes. Our results suggest that DHEA exerts its antioxidative effect by protecting the endogenous vitamin E of LDL.

Characterization of cDNAs encoding isoforms of hamster 3 beta-hydroxysteroid dehydrogenase/delta 5-->4 isomerase.

Western blot analyses of various hamster tissues reveal high levels of expression of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in adrenal and liver, and moderate levels of expression in kidney. The expression in liver is sexually dimorphic; very high levels of protein are observed in adult male liver but very low levels are seen in the female liver. Three distinct cDNAs encoding isoforms of 3 beta-HSD were isolated from hamster cDNA libraries. The type 1 isoform is a high-affinity dehydrogenase/isomerase expressed in adrenal and male kidney. The type 2 isoform is also a high-affinity dehydrogenase/isomerase expressed in kidney and male liver. The type 3 enzyme is a 3-ketosteroid reductase expressed predominantly in kidney. Sequencing of the clones showed that all three are structurally very similar, although types 1 and 2 share the greatest degree of similarity. Immunohistochemical staining for 3 beta-HSD in the adrenal was found throughout the adrenal cortex. In the kidney staining was confined to tubules, and in the liver, heavy staining was found in hepatocytes. The cloning of cDNAs for 3 beta-HSD from the liver and kidney should help in elucidating the function of this enzyme in these tissues.