Adrenodoxin mRNA Research Articles

Screening of ovarian steroidogenic pathway in Ciona intestinalis and its modulation after tributyltin exposure

In this study, we have identified several ovarian steroids in Ciona with high similarity to vertebrate steroids and showed that cholesterol, corticosterone, dehydroepiandrosterone, estrone, estradiol-17β, testosterone, pregnenolone, progesterone, have identical molecular spectra with vertebrate steroids. In addition, we have studied the effects of an endocrine disruptor (tributyltin: TBT) on these sex hormones and their precursors, ovarian morphology, and gene expression of some key enzymes in steroidogenic pathway in the ovary of Ciona. Ovarian specimens were cultured in vitro using different concentrations of TBT (10 − 5 , 10 − 4 and 10 − 3 M). Ethanol was used as solvent control. Gene expression analysis was performed for adrenodoxin (ADREN) and adrenodoxin reductase (ADOX) (mediators of acute steroidogenesis) and 17β-hydroxysteroid dehydrogenase (17β-HSD). These transcripts were detected and measured by quantitative (real-time) polymerase chain reaction (qPCR). Sex steroids and their precursors were identified and quantified by a gas chromatography-mass spectroscopy (GC-MS) method. Exposure of Ciona ovaries to TBT produced modulations (either increased or decreased) of sterols and sex steroid levels, whereas no significant differences in ADREN, ADOX or 17β-HSD mRNA expression patterns were observed. Histological analysis shows that TBT produced several modifications on Ciona ovarian morphology that includes irregular outline of nuclear membrane, less compacted cytoplasm, in addition to test and granulosa cells that were detached from the oocyte membrane. Given that the ascidians represent very simple experimental models for the study of endocrine disruption by environmental contaminants, our findings provide excellent models for multiple identification and quantification of sex steroid and their precursors in biological samples exposed to endocrine-disrupting chemicals and for direct extrapolation of such effects across taxonomic groups and phyla. In addition, these results suggest that C iona intestinalis may be a suitable species for molecular ecotoxicological studies and biomarker model for endocrine-disrupting effects in marine invertebrates.

Developmental expression of placental trophoblast P-450 cholesterolside-chain cleavage, adrenodoxin and Δ 5-3β-Hydroxysteroid dehydrogenase/isomerase messenger ribonucleic acids during baboon pregnancy

The present study determined whether the elevation in oestrogen, which occurs with advancing baboon pregnancy, is associatedwith a developmental increase in expression of the placental enzymes catalysing progesterone synthesis. The mRNA levels for P-450 cholesterol side-chain cleavage (P-450 scc), adrenodoxin, and Δ 5-3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) were assessed by Northern blot analysis in placental syncytiotrophoblasts isolated from baboons in early (days 58–65), mid- (days 97–113) and late (days 161–175), gestation (term=184 days). Placental villous tissue was dispersed and subjected to 50 per cent Percoll density gradient centrifugation to obtain primarily syncytiotrophoblasts. Mean ( ± s.e.) P-450 scc mRNA level, expressed as a ratio of β-actin in the syncytiotrophoblast-rich fraction, progressively increased with advancing pregnancy to a level in late gestation (1.81 ± 0.28 arbitrary units) that was approximately sixfold ( P<0.01) greater than in early gestation (0.31 ± 0.08) and approximately twofold greater ( P<0.05) than in mid-gestation (0.97 ± 0.24). In contrast adrenodoxin mRNA expression was similar at early (0.97), mid- (1.14 ± 0.12) and late (1.16 ± 0.13) gestation. Syncytiotrophoblast 3β-HSD mRNA levels also remained constant in early (1.69), mid- (1.89 0.41) and late (1.34 0.41) gestation. On the basis of these findings, we propose that villous syncytiotrophoblasts undergo functional/biochemical differentiation, resulting in a coordinated upregulation of specific components of the steroid biosynthetic pathway required for progesterone biosynthesis during the course of primate pregnancy.

Expression of the P450 side-chain cleavage and adrenodoxin genes begins during early stages of adrenal cortex development.

The steroid hormone products of the fetal adrenal cortex play an essential role in normal maturation of several organ systems during fetal development. In addition, adrenal steroids appear to play a local role in the establishment and maintenance of the chromaffin cells of the adrenal cortex. Despite these developmental roles of cortical steroids, little is known about when the cells of the fetal rat adrenal cortex begin to undergo biochemical differentiation into cells capable of producing steroid hormones and whether the timing of developmental changes in cortical properties is related to chromaffin cell differentiation. To investigate these problems, in situ hybridization and immunocytochemistry were used to examine the ontogeny of expression of both the P450 side-chain cleavage (P450scc) and adrenodoxin genes during rat development. Transcripts from both genes (but not P450c17) and the respective proteins encoded by them were detected specifically in the cells of the presumptive cortex as early as embryonic day 12 (e12), which is several days before the layered architecture of the adrenal cortex is established and the earliest age at which biochemical differentiation of these cells has been detected. The spatial and temporal expression patterns for both genes were similar over the period examined (e12-e16.5), and no heterogeneity of expression was observed among cortical cells. In addition, significant increases in the accumulation of P450scc and adrenodoxin mRNA transcripts occurred during the midgestational period, when the synthesis and secretion of ACTH from the fetal pituitary are increasing.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional activation of adrenocortical steroidogenic genes by high potassium or low sodium intake

We have previously shown that the long-term alterations in the intake of sodium and potassium which stimulated aldosterone production in the rat adrenal significantly increased cytochrome P450 scc (P450 scc) and P450 l11β (P450 11β) mRNA's and also the mRNA of their electron donor adrenodoxin. In the present study run-on analyses showed an accumulation of nascent RNA in isolated nuclei of zona glomerulosa cells in K +-supplemented and Na +-depleted rats for P450 c (5- and 6-fold), 3β-HSD (3.6- and 2.0-fold) and P450 11β (6.0- and 6.1-fold), but not for P450 c21 (1.4- and 1.1-fold). In contrast, that of adrenodoxin decreased (0.6-fold) in high K + and remained near control (1.3-fold) in low Na + intake. Moderate variations in the rate of transcription of P450 scc, P450 c21, P450 11β and adrenodoxin genes were observed in the zona fasciculata-reticularis cells of the treated rats. Our results thus demonstrated that positive modulators of aldosterone such as long-term K + supplementation and Na + restriction provoked an increase in transcription of the genes encoding key regulatory steroidogenic enzymes of aldosterone biosynthesis in the zona glomerulosa. The rates of transcription of the genes encoding 3β-HSD and P450 c21, two enzymes catalyzing intermediate steps in the aldosterone pathway, were moderately affected by such treatments. However, according to the known stimulation of adrenodoxin mRNA levels following these treatments, a decreased turnover of the adrenodoxin mRNA rather than initiation of transcription of its gene might be involved in the response to K + ions, and partially so in the response to Na + restriction. Finally, the effects of salt-modified intake were mainly restricted to the zona glomerulosa cells, which are solely responsible for aldosterone production.

Regulation of human cytochrome P450scc and adrenodoxin messenger ribonucleic acids in JEG-3 cytotrophoblast cells.

Cycloheximide generally inhibits steroidogenesis, but has different effects on the accumulation of the mRNAs for various steroidogenic enzymes in different species, tissues, and cell lines. In bovine adrenocortical cells, cycloheximide prevents ACTH- or cAMP-induced accumulation of the mRNAs for cytochrome P450scc and adrenodoxin, but in human cells, cycloheximide induces the accumulation of adrenodoxin mRNA. To study the potential role of the 3'-untranslated regions, and especially the AU-rich regions, of adrenodoxin and P450scc mRNAs in cycloheximide-sensitive regulation of mRNA accumulation, we constructed a series of vectors expressing P450scc or adrenodoxin mRNA with its own or each other's 3'-untranslated sequences and transfected them into human JEG-3 cytotrophoblast cells. Removal of the AU-rich 3'-untranslated sequences of adrenodoxin mRNA and replacing them with the 3'-untranslated region of P450scc did not alter the abundance or apparent stability of this mRNA, or its inducibility by cycloheximide or cAMP. Substituting the AU-rich 3'-untranslated region of adrenodoxin mRNA (which contains three copies of the AUUUA sequence) for the 3'-untranslated region of P450scc did not alter the inducibility of P450scc mRNA with forskolin. Inhibition of transcription with actinomycin-D elicited no difference in the adrenodoxin mRNA half-life in JEG-3 cells treated with forskolin, cycloheximide, or both. RNA polymerase run-on assays show little effect of forskolin on adrenodoxin gene transcription, while P450scc gene transcription was induced. These data suggest that the principal means for regulating P450scc mRNA is transcriptional, while the principal regulation of adrenodoxin is posttranscriptional. This posttranscriptional regulation of adrenodoxin mRNA is not mediated by the AUUUA sequences or other segments of the 3'-untranslated region.

Regulation of the cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin mRNAs in cultured choriocarcinoma cells

Cytochrome P-450 scc ( p450 scc) catalyzes the cholesterol side-chain cleavage reaction, a rate-limiting enzymatic step for progesterone synthesis in trophoblastic and other steroidogenic cells. Adrenodoxin is the iron/sulfur protein donating electrons to P-450 scc during this reaction. We examined the effects of cholera toxin (CT), an activator of adenylate cyclase, and 12- O-tetradecanoylphorbol acetate (TPA), a phorbol ester protein kinase C activator, on the levels of mRNAs encoding P-450 scc and adrenodoxin in JEG-3 choriocarcinoma cells. CT induced in a concentration- and time-dependent manner P-450 scc and adrenodoxin mRNA levels to 8-fold and 1.5-fold above that of control, respectively. TPA also increased P-450 scc and adrenodoxin mRNA levels about 3-fold and 1.5-fold above that of control, respectively. Epidermal growth factor (EGF) was found to weakly induce P-450 scc mRNA accumulation with a maximal 20% stimulation above basal levels. The effects of CT and TPA were apparently additive on both mRNAs. The protein synthesis inhibitor cycloheximide diminished basal, CT-, TPA-, and EGF-stimulated P-450 scc mRNA accumulation whereas the opposite was observed for the adrenodoxin mRNA. Insulin-like growth factor I (IGF-1) appeared to have no effect on either mRNA. These data indicate that: (1) the accumulation of P-450 scc and adrenodoxin mRNAs is mainly controlled by the cyclic adenosine 3′, 5′-monophosphate (cAMP)-dependent pathway but their stimulation by TPA- and EGF-induced signals may also play a weaker synergistic role; (2) the protein synthesis inhibitor cycloheximide inhibits basal, CT-, TPA- and EGF-stimulated P-450 scc mRNA levels while it increases the expression of adrenodoxin mRNA suggesting that in the malignant trophoblasts these two enzyme mRNAs are differentially controlled.

Expression and Regulation of Adrenodoxin and P450scc mRNA in Rodent Tissues

The rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone. This reaction occurs in steroidogenic tissue in the inner mitochondrial membrane, and is mediated by the cholesterol side-chain cleavage enzyme. This enzyme system transfers electrons from NADPH to cholesterol through its three protein components: adrenodoxin reductase, adrenodoxin, and the terminal oxidase, P450scc. We have previously shown that P450scc mRNA is regulated by tropic hormones and cAMP by a cycloheximide-independent mechanism in mouse Leydig tumor MA-10 cells. We now show that the mRNA for adrenodoxin, another component of the cholesterol side-chain cleavage enzyme system, is regulated by tropic hormones and cAMP in MA-10 cells. We cloned rat adrenodoxin cDNA to analyze adrenodoxin mRNA in various rat tissues and in MA-10 cells by RNase protection assays. Adrenodoxin mRNA is found in virtually all rat tissues examined, although it is most abundant in adrenals, ovaries, and testes. MA-10 cells synthesize two species of adrenodoxin mRNA, one of 1.2 kb and the other of 0.8 kb. Both of these adrenodoxin mRNAs are increased approximately six-fold by 1 mM 8-Br-cAMP, five-fold by 10 microM forskolin, and three-fold by both 25 ng/ml hCG and by 100 ng/ml LH. Maximal adrenodoxin mRNA accumulation occurs by 4 h of hormonal stimulation. The cAMP-mediated increase in adrenodoxin mRNA accumulation is independent of protein synthesis, since treatment with cycloheximide or puromycin in the absence or presence of cAMP does not inhibit, and even increases, adrenodoxin mRNA accumulation.

Regulation of rat adrenal messenger RNA and protein levels for cytochrome P-450s and adrenodoxin by dietary sodium depletion or potassium intake

The effect of low sodium and high potassium intake on rat adrenal zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR) were studied during a 7-day period, by analyzing mRNA and protein levels of various enzymes involved in aldosterone synthesis. In ZG significant increases in cytochrome P-450scc, P-450c21, P-450(11 beta), adrenodoxin mRNA and protein levels were observed after 2 days with either diet, and at day 7 these levels were further increased. The largest mRNA induction was observed at day 7 in sodium-depleted rats for P-450(11 beta), with a 4-fold increase, followed by 2.7- and 2.0-fold increases for P-450scc and P-450c21, respectively. A pattern similar to those of P-450scc and P-450(11 beta) was observed for adrenodoxin with a 2.1-fold increase after 7 days of Na+ restriction. In K(+)-loaded rats mRNA levels for P-450scc, P-450(11 beta), P-450c21, and adrenodoxin were also increased by 2.2-, 2.1-, 1.5-, and 1.9-fold respectively. Protein levels of these enzymes were also measured in ZG and showed increases similar to those of their respective mRNAs for both treatments. On the other hand, mRNA levels of P-450scc, P-450(11 beta), P-450c21, and adrenodoxin in ZFR were found significantly lower than in ZG, although they were slightly increased for both treated groups of rats as compared with controls. In addition, ZFR protein levels of corresponding enzymes did not fluctuate significantly under both ionic regimens. In conclusion, both low sodium and high potassium intakes act primarily on ZG. Their action on plasma aldosterone seems to be mediated by increasing both mRNA and protein and levels of steroidogenic enzymes, especially at the early step (cytochrome P-450scc) and even more at the late steps (cytochrome P-450(11 beta]. In addition, a close relationship appears to exist between the two mitochondrial P-450s and their electron donor adrenodoxin, since their mRNA and protein levels were similarly enhanced for both diets used.

Effects of 8-bromo-cAMP on expression of endocrine functions by cultured human trophoblast cells. Regulation of specific mRNAs

There is little information on the molecular events underlying the effects of cAMP on human chorionic gonadotropin (hCG) and particularly steroidal hormone production in normal trophoblasts. We examined the effects of 8-bromo-cAMP on mRNAs encoding two components of the cholesterol side-chain cleavage system, cytochrome P-450 scc and adrenodoxin, and the α and β subunits of hCG in cultured cytotrophoblasts. cAMP caused an increase in all of these mRNAs within 24 h, whereas actin mRNA declined. α-hCG mRNA increased first, followed by adrenodoxin, β hCG and cytochrome P-450 scc mRNAs.The effects of 8-bromo-cAMP on α- and β-hCG, adrenodoxin, and cytochrome P-450 scc mRNAs, in cytotrophoblasts and JEG-3 choriocarcinoma cells, required the catalytic unit of protein kinases since H-7, a kinase inhibitor, blocked the increase in the mRNAs and prevented the stimulation of hCG and progesterone secretion. 8-Bromo-cAMP promoted a rapid increase in α-hCG mRNA in cytotrophoblasts in the presence of cycloheximide, an inhibitor of protein synthesis. In cytotrophoblasts, cycloheximide reduced basal and 8-bromo-cAMP-stimulated adrenodoxin mRNA abundance. In contrast, basal and cAMP-stimulated adrenodoxin mRNA was augmented by cycloheximide in JEG-3 cells. These data indicate that: (1) cAMP regulates expression of endocrine functions of normal human trophoblast cells at least in part by controlling the abundance of mRNAs encoding steroidogenic enzymes and hCG subunits; (2) the actions of cAMP require the intermediacy of protein kinases; (3) there are differences in the sensitivity of the cAMP regulation of various mRNAs to inhibitors of protein synthesis; and (4) there are differences between normal and transformed trophoblasts with respect to the control of certain transcripts.

Effects of dietary sodium restriction and potassium intake on cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin mRNA levels.

We studied the effect of dietary sodium restriction (3 weeks) and high potassium intake (7 days) on transcriptional regulation of cytochrome P-450 cholesterol side chain cleavage (P-450 scc) and adrenodoxin (Adx) in rat adrenal glands. Northern blotting analysis demonstrated that both treatments markedly increased P-450scc and Adx mRNA levels in the zona glomerulosa (Z-G) and the zona fasciculata-reticularis (Z-F-R) compared with controls. The Z-G appears to be more sensitive to variations in electrolytes than does the Z-F-R. The low sodium diet provoked a 2.9-fold increase in P-450scc mRNA level in the Z-G compared to 2.1-fold in the Z-F-R, whereas Adx mRNA levels were enhanced 2.2- and 1.7-fold respectively in these two zones. Restriction of sodium intake provoked significant increases in plasma ACTH, aldosterone and corticosteroids compared with controls. In the Z-G of KCl-loaded rats, we found a 1.6-fold increase in P-450scc and a 2.1-fold increase in Adx mRNA levels, and in the Z-F-R there was a 1.7- and 1.8-fold enhancement. There were no changes in beta-actin mRNA levels upon dietary treatments. These results thus indicate that both sodium depletion and high potassium intake in rats could act at the transcriptional level of P-450scc and Adx, two components of a rate-limiting step in steroidogenesis leading to aldosterone production. In addition, the elevation in plasma ACTH level in response to Na+ restriction suggests a possible involvement of ACTH in the response of the adrenal glands to sodium depletion.

Transcription of the bovine adrenodoxin gene produces two species of mRNA of which only one is translated into adrenodoxin.

The 5'-end of the bovine adrenodoxin gene contains unique structural characteristics. Exons 1 and 2 appear to encode different presequences for this mitochondrial precursor protein. However, exon 1 contains a stop codon (TAA) in place of amino acid 15. Thus, while two species of bovine adrenodoxin mRNA arise from a single gene, only the abundant mRNA species (90%) is translated into the adrenodoxin precursor which contains the presequence encoded by exon 2. The minor mRNA species (10%) contains the sequence encoded by exon 1 and cannot be translated into an adrenodoxin precursor. Presumably the sequence within exon 2 is removed from this minor mRNA species by alternative splicing. Furthermore, the initiation of transcription of the major adrenodoxin mRNA (exon 2) lies within intron 1 of this unusual gene, while that for the other mRNA (exon 1) lies in the 5'-flanking region. Thus, the adrenodoxin gene is the first example of a gene encoding a mitochondrial protein which falls into the category of genes having alternate promoters utilizing a pattern of alternative splicing.

Human adrenodoxin: cloning of three cDNAs and cycloheximide enhancement in JEG-3 cells.

Adrenodoxin is an iron-sulfur protein serving as an electron transport intermediate for two mitochondrial steroidogenic cytochromes P450. We have cloned and sequenced three human adrenal adrenodoxin cDNAs. The longest 5'-untranslated region was 131 bases long, and the coding sequences, identical in all three clones, predict a preprotein of 180 amino acids. The 3'-untranslated regions were 235, 596, and 776 bases long due to the presence of alternate polyadenylation sites. RNA transfer blots showed multiple size species of adrenodoxin mRNA consistent with finding multiple polyadenylation sites. Similar sized cross-hybridizing RNA species are found abundantly in the adrenal and testis and to a lesser degree in RNA from human fetal brain, spleen, placenta, kidney, liver, and intestine, as well as in cultured fibroblasts, suggesting the same or a very similar iron-sulfur protein is found in mitochondria of nonsteroidogenic tissues. JEG-3 cells, a transformed progesterone-producing line of trophoblastic origin, accumulate mRNAs for cytochrome P450scc (the mitochondrial cholesterol side-chain cleavage enzyme), adrenodoxin, and the fos oncogene when stimulated with 8-bromo-cyclic AMP. Addition of actinomycin D to such cultures blocked cAMP-induced accumulation of mRNAs for cytochrome P450scc and adrenodoxin. Addition of cycloheximide or puromycin to such cultures substantially reduced basal levels and markedly attenuated the cAMP-induced accumulation of cytochrome P450scc mRNA, but augmented the accumulation of adrenodoxin and fos mRNAs in additive and multiplicative fashions, respectively. These data indicate that the cAMP-induced synthesis of the steroidogenic machinery is not wholly dependent on cycloheximide-sensitive protein mediators.

Hormonal and developmental regulation of adrenodoxin messenger ribonucleic acid in steroidogenic tissues.

Adrenodoxin is an iron-sulfur protein found in the mitochondria of steroidogenic tissues. It participates in steroidogenesis as an electron transport intermediate for mitochondrial cytochromes P450, including P450scc, the cholesterol side-chain cleavage enzyme. Using a human adrenodoxin cDNA probe recently cloned in our laboratory, we examined the distribution and hormonal regulation of adrenodoxin mRNA in a variety of steroidogenic tissues. Adrenodoxin mRNA was found in all steroidogenic tissues examined. In human fetal testes, adrenodoxin mRNA was more abundant in early gestation, diminishing toward midterm in a pattern closely similar to that we reported previously for P450scc. Unlike P450scc, however, significant amounts of adrenodoxin mRNA were detected in human fetal ovaries, with no discernible gestation-dependent change. The abundance of adrenodoxin mRNA was increased in cultured human granulosa cells by treatment with hCG, FSH, cAMP, and cholera toxin. In human fetal adrenal cells, ACTH and cAMP stimulated accumulation of adrenodoxin mRNA, while in cultured human fetal testicular cells and cultured fetal rhesus monkey ovarian cells, both hCG and cAMP stimulated accumulation of adrenodoxin mRNA. In all of these systems, the accumulation of adrenodoxin mRNA closely paralleled the response of P450scc. These data suggest that the genes for these functionally related but structurally unrelated proteins are regulated in a coordinate manner.

Multiple species of bovine adrenodoxin mRNA. Occurrence of two different mitochondrial precursor sequences associated with the same mature sequence.

Bovine adrenodoxin mRNA is found to consist of several distinct mRNA species which can be divided into two sets. Each set utilizes at least three of four separate poly(A)+ addition sites providing an explanation of the three sizes of adrenodoxin mRNA (1.75, 1.4, and 0.95 kilobases) previously observed in bovine adrenocortical RNA by this laboratory (Okamura, T., John, M.E., Zuber, M.X., Simpson, E.R., and Waterman, M.R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5705-5709). The two sets are distinguished from one another by a unique 5' sequence leading to two different amino acid sequences (approximately 10% homology) for the precursor portion of this nuclear encoded, mitochondrial protein. A common mature adrenodoxin sequence is encoded by both sets of mRNA. One set of RNAs is 10-fold more abundant than the other, but the levels of both sets can be induced by treatment of primary bovine adrenocortical cell cultures with adrenocorticotropin. The biological significance of these two types of adrenodoxin precursor sequences remains obscure.

Molecular cloning and amino acid sequence of the precursor form of bovine adrenodoxin: evidence for a previously unidentified COOH-terminal peptide.

Several recombinant cDNA clones specific for the mitochondrial iron-sulfur protein adrenodoxin have been identified in a bovine adrenocortical cDNA library. One clone (pBAdx4) contains a 900-base-pair insert that includes the entire amino acid coding region of the adrenodoxin precursor protein. The amino acid sequence of mature adrenodoxin deduced from the nucleotide sequence of pBAdx4 is identical with that determined by protein sequencing except for three amide changes. The previously undetermined sequence of the adrenodoxin NH2-terminal precursor segment (58 amino acids) contains several basic residues, a characteristic feature of the precursor segment of proteins destined for mitochondria. In addition, a 14 amino acid extension is present at the COOH terminus of the mature adrenodoxin sequence. Whether this represents a COOH-terminal precursor segment is not clear. Three different adrenodoxin mRNAs are present [1.75, 1.4, and 0.95 kilobase(s) long] in bovine adrenocortical RNA. RNA from bovine corpus luteum, liver, and kidney contains transcripts that hybridize to adrenodoxin cDNA. Accumulation of adrenodoxin mRNA occurs in cultured bovine adrenocortical cells after treatment with ACTH or dibutyryl-cAMP, similar to that observed for the mitochondrial steroid hydroxylases that it services--namely, the cholesterol side-chain-cleavage cytochrome P-450 and the steroid 11 beta-hydroxylase cytochrome P-450.