Biomedical subjects

N A Compagnone

Publications and source records attributed to N A Compagnone.

11 recordsLinked to original sources

PubMed2001

Modulation of steroidogenesis by selenium in a novel adrenal cell line developed using targeted tumorigenesis.

UNLABELLED: Glutathione peroxidase (GPx-1) is a selenoenzyme that metabolizes H(2)O(2), a source of potentially toxic free radicals. Steroidogenesis is markedly inhibited by H(2)O(2) in vitro. OBJECTIVE: to study the effects of selenium deficiency on GPx activity and adrenal steroidogenesis in a novel adrenal cell line developed using targeted tumorigenesis. METHODS: AN4Rppc7 cells were grown for 7 days in serum-free medium. 8-Br-cAMP-stimulated concentrations of steroid hormones were measured by RIA. StAR (Steroid Acute Reactive Protein) mRNA was measured by Northern blot. RESULTS: selenium deficiency caused a 99% There was a 51%, progesterone, corticosterone and aldosterone production, respectively (p<0.05 by ANOVA). StAR mRNA was not affected by selenium. CONCLUSIONS: selenium deficiency causes a marked decrease in GPx activity. Decreased steroid hormone production occurs for selenium concentrations equal or lower than 5 nM. The absence of changes in StAR mRNA content suggests that selenium deficiency does not affect cholesterol access to the mitochondria.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2001

Biosynthesis and action of neurosteroids.

Over the past decade, it has become clear that the brain, like the gonad, adrenal and placenta, is a steroidogenic organ. However, unlike classic steroidogenic tissues, the synthesis of steroids in the nervous system requires the coordinate expression and regulation of the genes encoding the steroidogenic enzymes in several different cell types (neurons and glia) at different locations in the nervous system, and at distances from the cell bodies. The steroids synthesized by the brain and nervous system, given the name neurosteroids, have a wide variety of diverse functions. In general, they mediate their actions, not through classic steroid hormone nuclear receptors, but through other mechanisms such as through ion gated neurotransmitter receptors, or through direct or indirect modulation of other neurotransmitter receptors. We have briefly summarized the biochemistry of the enzymes involved in the biosynthesis of neurosteroids, their localization during development and in the adult, and the regulation of their expression, highlighting both similarities and differences between expression in the brain and in classic steroidogenic tissues.

Animals↗

PubMed2001

Developmental gonadal expression of the transcription factor SET and its target gene, P450c17 (17alpha-hydroxylase/c17,20 lyase).

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase/17,20 lyase activity needed for sex steroid synthesis. We recently characterized the nuclear phosphoprotein SET as a novel transcriptional regulator that binds to the -447/-399 region of the rat P450c17 gene, along with the transcription factors COUP-TF II, NGF-IB, and SF-1. Gel shift studies localized SET binding to nucleotides -410/-402. We have shown that SET activates transcription of the rat P450c17 gene in neuronal precursor cells and now show that it also activates transcription from the -418/-399 region of the rat P450c17 gene in mouse Leydig MA-10 cells. Studying the ontogenic expression of SET and P450c17 in the rodent gonad, we found that SET expression preceded P450c17 expression in the embryonic genital ridge, suggesting that SET may be important for initiating P450c17 expression in this region. Expression of SET also preceded P450c17 expression in the testis and ovary, and its expression was much greater during embryogenesis than in the adult gonad. In the adult rat testis, P450c17 was expressed only in Leydig cells, while SET was expressed in Leydig cells and in spermatocytes. In the adult rat ovary, P450c17 was expressed only in theca cells, while SET was expressed in theca cells and also in oocytes. Because SET is expressed early in development in the genital ridge and in the testis and ovary, and because SET has many functions in addition to its activity as a transcription factor, we determined whether SET acts a transcription factor in oocytes. The SET protein was detected by Western blots in Xenopus oocytes from stages II through VI and in mature oocytes. Using extracts of Xenopus oocytes in gel shift assays, we detected a protein that bound to the -418/-399 region of the rat P450c17 gene, to which SET binds. Nuclear injection of either a -418/-399TK32LUC wildtype reporter construct or a construct containing a mutant SET site into Xenopus oocytes from stages III through VI resulted in activation of luciferase activity with the wildtype but not the mutant construct in all stages. These data suggest that Xenopus SET is able to bind to specific DNA sequences to activate transcription at all stages of Xenopus oogenesis. These data indicate that SET is an evolutionarily conserved transcription factor that participates in the early ontogenesis of the gonadal system, regulates P450c17 gene transcription in Leydig cells, and may also activate other genes expressed in immature oocytes, thus playing a role in oocyte development.

Animals↗

PubMed2000

Neurosteroids: biosynthesis and function of these novel neuromodulators.

Over the past decade, it has become clear that the brain is a steroidogenic organ. The steroids synthesized by the brain and nervous system, given the name neurosteroids, have a wide variety of diverse functions. In general, they mediate their actions, not through classic steroid hormone nuclear receptors, but through ion-gated neurotransmitter receptors. This paper summarizes what is known about the biosynthesis of neurosteroids, the enzymes mediating these reactions, their localization during development and in the adult, and their function and mechanisms of action in the developing and adult central and peripheral nervous systems. The expression of the steroidogenic enzymes is developmentally regulated, with some enzymes being expressed only during development, while others are expressed during development and in the adult. These enzymes are expressed in both neurons and glia, suggesting that these two cell types must work in concert to produce the appropriate active neurosteroid. The functions attributed to specific neurosteroids include modulation of GABA(A) and NMDA function, modulation of sigma receptor function, regulation of myelinization, neuroprotection, and growth of axons and dendrites. Neurosteroids have also been shown to modulate expression of particular subunits of GABA(A) and NMDA receptors, providing additional sites at which these compounds can regulate neural function. The pharmacological properties of specific neurosteroids are described, and potential uses of neurosteroids in specific neuropathologies and during normal aging in humans are also discussed.

Animals↗

PubMed2000

Novel role for the nuclear phosphoprotein SET in transcriptional activation of P450c17 and initiation of neurosteroidogenesis.

Neurosteroids are important endogenous regulators of gamma-aminobutryic acid (GABA(A)) and N-methyl-D-aspartate (NMDA) receptors and also influence neuronal morphology and function. Neurosteroids are produced in the brain using many of the same enzymes found in the adrenal and gonad. The crucial enzyme for the synthesis of DHEA (dehydroepiandrosterone) in the brain is cytochrome P450c17. The transcriptional strategy for the expression of P450c17 is clearly different in the brain from that in the adrenal or gonad. We previously characterized a novel transcriptional regulator from Leydig MA-10 cells, termed StF-IT-1, that binds at bases -447/-399 of the rat P450c17 promoter, along with the known transcription factors COUP-TF (chicken ovalbumin upstream promoter transcription factor), NGF-IB (nerve growth factor inducible protein B), and SF-1 (steroidogenic factor-1). We have now purified and sequenced this protein from immature porcine testes, identifying it as the nuclear phosphoprotein SET; a role for SET in transcription was not established previously. Binding of bacterially expressed human and rat SET to the DNA site at -418/-399 of the rat P450c17 gene transactivates P450c17 in neuronal and in testicular Leydig cells. We also found SET expressed in human NT2 neuronal precursor cells, implicating a role in neurosteroidogenesis. Immunocytochemistry and in situ hybridization in the mouse fetus show that the ontogeny and distribution of SET in the developing nervous system are consistent with SET being crucial for initiating P450c17 transcription. SET's developmental pattern of expression suggests it may participate in the early ontogenesis of the nervous, as well as the skeletal and hematopoietic, systems. These studies delineate an important new factor in the transcriptional regulation of P450c17 and consequently, in the production of DHEA and sex steroids.

Amino Acid Sequence↗

PubMed1998

Dehydroepiandrosterone: a potential signalling molecule for neocortical organization during development.

Dehydroepiandrosterone (DHEA) and its sulfate derivative (DHEAS) are the most abundant steroids produced by the human adrenal, but no receptors have been identified for these steroids, and no function for them has been established, other than as precursors for sex steroid synthesis. DHEA and DHEAS are found in brains from many species, and we have shown that enzymes crucial for their synthesis, especially P450c17 (17alpha-hydroxylase/c17,20 lyase), are expressed in a developmentally regulated, region-specific fashion in the developing rodent brain. One region of embryonic expression of P450c17, the neocortical subplate, has been postulated to play a role in guiding cortical projections to their appropriate targets. We therefore determined if products of P450c17 activity, DHEA and DHEAS, regulated the motility and/or growth of neocortical neurons. In primary cultures of mouse embryonic neocortical neurons, DHEA increased the length of neurites containing the axonal marker Tau-1, and the incidence of varicosities and basket-like process formations in a dose-dependent fashion. These effects could be seen at concentrations normally found in the brain. By contrast, DHEAS had no effect on Tau-1 axonal neurites but increased the length of neurites containing the dendritic marker microtubule-associated protein-2. DHEA rapidly increased free intracellular calcium via activation of N-methyl-D-aspartate (NMDA) receptors. These studies provide evidence of mechanisms by which DHEA and DHEAS exert biological actions, show that they have specific functions other than as sex steroid precursors, mediate their effects via non-classic steroid hormone receptors, and suggest that their developmentally regulated synthesis in vivo may play crucial and different roles in organizing the neocortex.

2-Amino-5-phosphonovalerate↗

PubMed1997

Characterization of adrenocortical cell lines produced by genetically targeted tumorigenesis in transgenic mice.

Using transgenic mice, we targeted SV40 T antigen and the bacterial neomycin resistance gene to steroidogenic tissues using a human P450 cholesterol side-chain cleavage promoter. Expression of SV40 T antigen resulted in adrenocortical tumors. Adrenocortical cell lines from one of these tumors (ST5R) was previously characterized. We have now obtained clonal lines from the second more differentiated tumor. After dispersion of the left adrenal tumor, ST5L parental cells were selected with G418 and subcloned. The resulting adrenocortical subcloned cell lines are more highly differentiated than those cell lines resulting from the right adrenal tumor (ST5R). ST5L cell lines secrete progesterone and corticosterone to varying degrees, whereas ST5R cells secrete only progesterone. One of the clonal cell lines, ST5Lc16, expresses both P450c11 beta and P450c11AS mRNAs, which normally are regionally distributed in different zones of the adrenal cortex. Thus, ST5Lc16 cells may be progenitor cells for both glomerulosa and fasciculata cells and may provide clues to the cellular and molecular events leading to the differentiation of the glomerulosa and the fasciculata-reticularis. Other ST5Lc cell lines are more representative of the fasciculata-reticularis, because they express P450c11 beta mRNA and secrete corticosterone, and they neither express P450c11AS mRNA nor do they secrete aldosterone. All cell lines also have 21-hydroxylase activity, but none express P450c21, indicating that some other, as yet unidentified, enzyme has this activity. In all cell lines, steroid secretion is regulable by cAMP stimulation but not by ACTH stimulation. All ST5L cell lines also express mouse renin-1 mRNA. In addition to their utility in studies of adrenal steroidogenesis, these cell lines may also be useful in studying the etiology of adrenocortical tumors.

Adrenal Cortex↗

PubMed1997

Expression of steroid sulfatase during embryogenesis.

Neurosteroids are steroids that are synthesized de novo in the brain from cholesterol and, in general, mediate their effects through ion-gated channel receptors such as gamma-aminobutyric acidA (GABA[A]) and N-methyl-D-aspartate receptors rather than through classical nuclear steroid hormone receptors. Steroid hormones are known to exist not only as free compounds, but also as sulfated derivatives. Pharmacological studies indicate that unconjugated and sulfated steroids, such as pregnenolone and pregnenolone sulfate, may have opposite effects on GABA(A) receptors. Thus, pregnenolone acts as a potent positive allosteric modulator of gamma-aminobutyric acid action at GABA(A )receptors, whereas pregnenolone sulfate acts as a potent negative modulator. Recent experiments also suggest that dehydroepiandrosterone and dehydroepiandrosterone sulfate may have distinct effects on growth of neurites from embryonic neocortical neurons in vitro. Thus, regulation of steroid sulfation may have profound behavioral and morphological effects on the nervous system. We, therefore, studied the developmental expression of the enzyme steroid sulfatase (STS), which converts sulfated steroids to free steroids. By in situ hybridization, STS messenger RNA was expressed in the embryonic mouse cortex, hindbrain, and thalamus during the last third of gestation. The sites of expression of STS were similar to those of P450c17, suggesting that these two enzymes may have concerted actions in similar functional processes.

Animals↗

PubMed1995

Steroidogenic enzyme P450c17 is expressed in the embryonic central nervous system.

Neurosteroids are steroids that are synthesized de novo in the brain and include some classical (adrenal and gonadal steroids) and some unique brain-specific steroids. Neurosteroids are thought to mediate their action through ion gated channel receptors such as gamma-aminobutyric acid(A) and N-methyl-D-aspartate rather than through classical nuclear steroid hormone receptors. Some enzymes involved in neurosteroidogenesis have been identified as those found in steroidogenic tissues, and some may be unique to the brain. We previously demonstrated that the messenger RNAs (mRNA) for the cholesterol side-chain cleavage enzyme, cytochrome P450scc, and one form of 11 beta-hydroxylase, cytochrome P450c11 beta, are regionally expressed in the adult rat brain. However, cytochrome P450c17, which has 17-hydroxylase and 17,20-lyase activity and is thought to be required for the synthesis of dehydroepiandrosterone, was not detected in any region of the rat brain, even though dehydroepiandrosterone is one of the most abundant neuroactive steroids. We now demonstrate that P450c17 is expressed in the nervous system of the developing rodent embryo. By ribonuclease protection assays, P450c17 mRNA was found in the trunk but not in the head of rat embryos but reverse transcriptase-polymerase chain reaction analysis showed expression of P450c17 mRNA in the head of E15.5 to E19.5 rat embryos. Immunocytochemically detectable P450c17 protein was expressed in the nervous system as early as embryonic day E10.5 in the mouse, mainly in tissue derived from the neural crest. Neuronal cell bodies as well as fibers staining for P450c17 were observed in the central and peripheral nervous systems. The sites of P450c17 expression in the peripheral nervous system suggest it may be involved in a wide variety of sensory-motor functions. In the central nervous system, cell bodies expressing P450c17 are found in the hind brain, in mesencephalic nuclei, and in a region in the location of the locus coeruleus, but in cells distinct from those expressing the dopamine-beta-hydroxylase. Furthermore, its particular location and temporal expression in axons reaching the cortical areas suggest it is a marker for the axonal growth in this region, and that its neurosteroid product may be a signal for targeting cortical axons during embryogenesis.

Animals↗

PubMed1995

Expression of the steroidogenic enzyme P450scc in the central and peripheral nervous systems during rodent embryogenesis.

Neurosteroids are steroids that are synthesized de novo in the brain and include some classical (adrenal and gonadal) steroids and some unique brain-specific steroids. Neurosteroids are thought to mediate their action through ion-gated channel receptors, such as gamma-aminobutyric acid(A) and N-methyl-D-aspartate rather than through classical nuclear steroid hormone receptors. Some enzymes involved in neurosteroidogenesis have been identified as those found in steroidogenic tissues, and some may be unique to the brain. We previously demonstrated that the messenger RNAs for the cholesterol side-chain cleavage enzyme, P450scc, and one form of 11 beta-hydroxylase, P450c11 beta, are regionally expressed in the adult rat brain. We now demonstrate that P450scc is expressed in the nervous system of the developing rodent embryo in cell lineages derived from the neural crest. Despite the presence of readily detectable P450scc protein, a ribonuclease protection assay detected P450scc messenger RNA only in the trunks and not in the heads of male and female rat embryos. P450scc immunoreactive protein is continuously expressed in the central and peripheral nervous systems from embryonic day 9.5 in the rat. The sites of expression of P450scc are located mainly in sensory structures of the peripheral nervous system during embryogenesis, suggesting a possible function in coordinating environmental cues and behavior and in the development and organization of the nervous system.

Animals↗

PubMedDate not supplied

Orphan receptors, proto-oncogenes and other nuclear factors regulate P450C17 gene transcription.

Concerted, regulated expression of the rat P450c17 gene relies on the combined participation of multiple DNA regions and factors that bind to these sequences. SF-1 binds to at least two different regions, and has activities dependent upon the DNA context. COUP-TF, NGF-IB, together with SF-1, bind to overlapping regions, and the interaction among these factors increases or decreases transcription. A newly identified factor, the proto-oncogene SET, binds to a portion of the COUP-TF site, and strongly activates P450c17 transcription. SET mRNA is highly expressed early during development, and its expression decreases thereafter. P450c17 expression is not restricted to steroidogenic tissues, but rather is highly expressed in specific locations in the central (CNS) and peripheral nervous systems (PNS) during development, where its steroidal products may regulate neuronal maturation. We have shown that physiologic concentrations of steroids derived from P450c17 activity, DHEA and DHEAS, have direct and distinct effects on neocortical axonal and dendritic growth and differentiation. In the CNS and PNS, SET mRNA is expressed in regions where we previously reported P450c17 expression. The ontogeny of SET expression in CNS and PNS tissues as well as in steroidogenic tissues precedes P450c17 expression, suggesting that in addition to regulating P450c17 gene transcription in adrenals and gonads, SET may be crucial for the regulation of P450c17 gene transcription in the CNS and PNS as well.

Adrenal Cortex Neoplasms↗