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S H Mellon

Publications and source records attributed to S H Mellon.

At least 19 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 of neurosteroids and regulation of their synthesis.

The brain, like the gonads, adrenal glands, and placenta, is a steroidogenic organ. The steroids synthesized by the brain and by the 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 chapter summarizes the biochemistry of the enzymes involved in the biosynthesis of neurosteroids, their localization during development and in adulthood, and the regulation of their expression, highlighting both similarities and differences between expression in the brain and in classic steroidogenic tissues.

Animals↗
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↗
PubMed2001

Biosynthesis of the neurosteroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha hp), a specific inhibitor of FSH release.

The gonadal steroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP) is a neuroactive steroid with anxiolytic and analgesic actions. In addition, 3 alpha HP has been shown to inhibit GnRH activity on gonadotropes and selectively suppress FSH release from pituitary cells, without an effect on LH. The enzyme 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) has been presumed to be the enzyme responsible for the conversion of progesterone to 3 alpha HP, but this has never been confirmed in vitro or in vivo. We have now determined the mechanism of 3 alpha HP synthesis in vivo using specific enzyme inhibitors and in vitro using recombinant proteins. Incubation of [(3)H]progesterone with purified recombinant rat and human 3 alpha HSD isoforms showed that both the rat 3 alpha HSD and the human type 2(brain) 3 alpha HSD converted progesterone to 3 alpha HP. Age-dependent 3 alpha HP production was demonstrated in pituitary and cortex. Incubation of both tissues with indomethacin, a known 3 alpha HSD inhibitor, decreased the conversion of progesterone to 3 alpha HP by at least 70%, indicating that 3 alpha HSD was responsible for this conversion. As human type 2 3 alpha HSD is expressed in a region-specific fashion in the brain, 3 alpha HP may only be made in specific regions of the brain. Furthermore, the data suggest that the pituitary has the capacity for 3 alpha HP production, which may provide an additional mechanism for regulation of GnRH action.

20-alpha-Dihydroprogesterone↗
PubMed2001

Regulation of uterine gamma-aminobutyric acid(A) receptor subunit expression throughout pregnancy.

Uterine contractions at parturition depend upon a variety of factors, including gamma-aminobutyric acid (GABA)-ergic stimulation. A new subunit of the GABA(A) receptor, pi, has recently been identified as being particularly abundant in the rat uterus. Reduced derivatives of progesterone, such as the 3alpha,5alpha-reduced derivative termed allopregnanolone, modulate GABA(A) receptor activity and neuronal inhibition by modulating the frequency and duration of GABA(A) channel opening. This modulation depends on the specific subunit composition of the GABA(A) receptor. In particular, assembly of recombinant pi and delta GABA(A) receptor subunits into a functional GABA(A) receptor have been reported to reduce sensitivity to allopregnanolone. As allopregnanolone works through the GABA(A) receptor to reduce uterine contraction, we hypothesized that incorporation of the pi-subunit into this receptor in the uterus might change the sensitivity of the GABA(A) receptor to allopregnanolone and modulate parturition. We therefore determined the expression of GABA(A) receptor subunit messenger RNAs (mRNAs) in rat uteri from various gestational ages and determined the physiological properties of the receptors. GABA(A) pi-subunit mRNA abundance was constant throughout gestation, but decreased at the onset of labor. Other GABA(A) subunits fluctuated differently during pregnancy: GABA(A) alpha(1)-subunit mRNA expression increased, whereas alpha(2)- and delta-subunit mRNA expression decreased during pregnancy, and beta(3)-subunit mRNA only appeared on postpartum day 1. We determined how allopregnanolone affected the binding of muscimol, a ligand for the GABA(A) receptor, to rat uterine GABA(A) receptors throughout pregnancy. Allopregnanolone caused the greatest increase in muscimol binding to uterine GABA(A) receptors at 19.5 days gestation and the least increase during labor, a time when pi and alpha(1) receptor subunit mRNA concentrations were low, and delta and alpha(2) receptor subunit mRNA concentrations were high. Thus, the subunit composition of the GABA(A) receptor differs in rat uteri throughout gestation. These changes may also affect the sensitivity of the GABA(A) receptor to allopregnanolone and thus contribute to the regulation of parturition.

3-Hydroxysteroid Dehydrogenases↗
PubMed2000

Opposing early inhibitory and late stimulatory effects of insulin-like growth factor-I on myogenin gene transcription.

Insulinlike growth factors (IGFs) stimulate skeletal muscle cell differentiation in association with an increase in the mRNA of myogenin, a member of the MyoD family of skeletal muscle-specific transcription factors that plays an essential role in the differentiation process. However, this is a relatively late effect, requiring treatment periods of >24 h. In contrast, IGFs initially inhibit skeletal muscle cell differentiation, associated with a marked reduction in myogenin mRNA. The mechanisms by which IGF-I initially inhibits and subsequently stimulates myogenin expression are unknown. In the first 24 h, we find that IGF-I inhibits myogenin gene transcription by >80% but has no effect on myogenin mRNA stability. Similarly, in the first 24 h, IGF-I markedly inhibits myogenin promoter activity; the sequence -145 to -9 of the myogenin gene is sufficient to confer this inhibitory effect of IGF-I. In contrast, 48 h of treatment with IGF-I results in an increase in myogenin promoter activity that parallels the increase in myogenin steady-state mRNA. This increase in promoter activity is completely prevented in constructs lacking the sequence -1,565 to -375 of the myogenin gene. These data indicate that the early inhibitory and late stimulatory effects of IGF-I on myogenin expression are mediated at the level of transcription, and that these time-dependent, opposing effects of IGF-I on myogenin transcription are mediated by distinct regions of the myogenin gene. To our knowledge, this is the first demonstration of a gene whose promoter activity is initially inhibited and subsequently stimulated by IGF-I.

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↗
PubMed2000

Autocrine and paracrine Müllerian inhibiting substance hormone signaling in reproduction.

Members of the transforming growth factor beta (TGFbeta) superfamily are polypeptide growth factors that exhibit diverse effects on normal cell growth, adhesion, mesenchymal-epithelial interactions, cell differentiation, and programmed cell death. This chapter will discuss the work of ourselves and others on one member of this large superfamily, Müllerian inhibiting substance (MIS, or anti-Müllerian hormone, AMH) and its role in reproductive tract development and the adult gonad. Using recombinant MIS protein, it is possible to begin unraveling the molecular mechanism of duct involution in the embryo. Our recent results suggest that MIS triggers cell death by altering mesenchymal-epithelial interactions. In addition to the developmental effects of MIS in secondary sexual differentiation, expression studies of the MIS ligand and the MIS type II receptor (MISIIR) suggest a potential regulatory role for MIS in adult germ cell maturation and gonadal function. Recent data from others suggest that MIS may act in a paracrine manner to block differentiation of interstitial cells of the adult gonad by repressing all or some steps of steroidogenesis. Our studies are highly suggestive of direct repression of steroidogenic enzyme gene expression by activation of the MIS signaling pathway. Thus, for the first time, an opportunity to define fully target genes and components of the MIS signaling pathway may be possible.

Animals↗
PubMed1999

Selective serotonin reuptake inhibitors directly alter activity of neurosteroidogenic enzymes.

The neurosteroid 3alpha-hydroxysteroid-5alpha-pregnan-20-one (allopregnanolone) acts as a positive allosteric modulator of gamma-aminobutyric acid at gamma-aminobutyric acid type A receptors and hence is a powerful anxiolytic, anticonvulsant, and anesthetic agent. Allopregnanolone is synthesized from progesterone by reduction to 5alpha-dihydroprogesterone, mediated by 5alpha-reductase, and by reduction to allopregnanolone, mediated by 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD). Previous reports suggested that some selective serotonin reuptake inhibitors (SSRIs) could alter concentrations of allopregnanolone in human cerebral spinal fluid and in rat brain sections. We determined whether SSRIs directly altered the activities of either 5alpha-reductase or 3alpha-HSD, using an in vitro system containing purified recombinant proteins. Although rats appear to express a single 3alpha-HSD isoform, the human brain contains several isoforms of this enzyme, including a new isoform we cloned from human fetal brains. Our results indicate that the SSRIs fluoxetine, sertraline, and paroxetine decrease the K(m) of the conversion of 5alpha-dihydroprogesterone to allopregnanolone by human 3alpha-HSD type III 10- to 30-fold. Only sertraline inhibited the reverse oxidative reaction. SSRIs also affected conversions of androgens to 3alpha- and 3alpha, 17beta-reduced or -oxidized androgens mediated by 3alpha-HSD type II(Brain). Another antidepressant, imipramine, was without any effect on allopregnanolone or androstanediol production. The region-specific expression of 3alpha-HSD type II(Brain) and 3alpha-HSD type III mRNAs suggest that SSRIs will affect neurosteroid production in a region-specific manner. Our results may thus help explain the rapid alleviation of the anxiety and dysphoria associated with late luteal phase dysphoria disorder and major unipolar depression by these SSRIs.

3-Hydroxysteroid Dehydrogenases↗
PubMed1999

Ku autoimmune antigen is involved in placental regulation of rat P450c17 gene transcription.

The steroidogenic enzyme P450c17 (17alpha hydroxylase/C17,20 lyase) regulates a key branchpoint in steroidogenesis, as its activity directs the steroid biosynthetic pathways toward glucocorticoid or sex hormone synthesis. Expression of the P450c17 gene is transcriptionally regulated in steroidogenic tissues by cAMP. We showed that DNA between -84 and -55 in the rat P450c17 gene was bound uniquely by steroidogenic factor-1 (SF-1), which regulated both basal and cAMP-stimulated transcription in mouse adrenocortical and Leydig cells. SF-1 gene ablation experiments in mice indicate that SF-1 is not mandatory for placental steroidogenesis. We studied P450c17 gene regulation in the placenta using human placental JEG-3 trophoblast cells. Transfection of reporter luciferase gene constructs containing serial deletions of the 5' flanking region of the rat P450c17 gene showed that DNA between -98 and +13 mediated basal and cAMP-regulated transcription in placental JEG-3 cells, as it did in adrenal and Leydig cells. DNase footprints further identified a region between -88 and the TATA box that was bound by protein. Transfection of luciferase reporter constructs containing -84 to -55 of the rat P450c17 DNA ligated to the minimal promoter of the thymidine kinase gene showed that this DNA increased both basal and cAMP-simulated luciferase activity. Gel mobility shift assays identified two DNA-protein complexes with JEG-3 cell nuclear extracts that were different from complexes formed with MA-10 cell extracts and did not involve SF-1. Mutational analysis of the -84/-55 DNA showed that JEG-3 nuclear proteins bound to a site containing, but not identical to, the SF-1 sequence. One complex involved Ku autoimmune antigen, which bound to DNA sequence specifically. Overexpression of Ku antigen in MA-10 cells stimulated rat P450c17 gene transcription, thus demonstrating a biologic effect of Ku. Ku also bound to a similar region of the human P450c17 gene, and the DNA region to which Ku bound was transcriptionally active in JEG-3 cells. Ku was also found in extracts from rat placenta and bound to the -84/-55 rat P450c17 DNA. These data demonstrate a role of Ku in regulating P450c17 gene expression. These data further indicate that although human P450c17 is not normally expressed in the placenta, factors that could activate this gene are indeed present.

Adrenal Glands↗
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↗
PubMed1998

Polyribonucleotide phosphorylase is a double-stranded DNA-binding protein.

Polyribonucleotide phosphorylase (PNPase) is one of the critical components of the E. coli RNA degradosome, which consists of both PNPase and endoribonuclease RNase E. The function of this complex is to control the rate of mRNA degradation. The PNPase possesses two enzymatic activities, namely 3'-5' processive exoribonuclease activity and 5'-3' RNA polymerase activity. In the present study, we used conventional chromatography to purify an E. coli protein that binds to a specific double-stranded DNA sequence. Microsequencing of the purified protein showed that this DNA-binding protein was PNPase. Our data further demonstrate that PNPase binds to DNA in a sequence-specific manner. These data suggest that PNPase may have previously unappreciated DNA-related functions in addition to its known role in mRNA degradation.

Amino Acid Sequence↗
PubMed1998

25-Hydroxycholesterol is not a ligand for the orphan nuclear receptor steroidogenic factor-1 (SF-1).

The orphan nuclear receptor steroidogenic factor-1 (SF-1) is involved in the transcriptional regulation of all the steroid hydroxylase genes, and also regulates the transcription of the genes for Müllerian Inhibitory substance (MIS), alpha subunit of glycoprotein hormone, LHbeta, oxytocin, GnRH receptor, ACTH receptor, prolactin receptor, DAX-1, and steroidogenic acute regulatory protein. Other members of the nuclear receptor gene family, including steroid hormone, thyroid hormone, retinoic acid, PPAR, and vitamin D receptors must bind ligand to activate transcription, but SF-1 has been considered to be an orphan nuclear receptor because, when identified, it had no known ligand. A recent publication suggested that transcriptional regulation by SF-1, expressed in a non-steroidogenic CV-1 cells, could be activated by oxysterols suggesting that these compounds could serve as natural ligands for SF-1. We now demonstrate that 25-hydroxycholesterol, either added exogenously or synthesized endogenously in steroidogenic mouse Leydig MA-10 cells, did not act as a ligand for SF-1, as it did not increase transcription from six different SF-1-dependent DNA sequences. Furthermore, the abundance of these oxysterols in MA-10 cells was much less than concentrations needed for activation of SF-1 in CV-1 cells, indicating that SF-1 is not constitutively bound by ligand in MA-10 cells. Thus, in steroidogenic cells, transcriptional regulation of the steroid hydroxylase genes by SF-1 does not depend upon the presence of 25-hydroxycholesterol, and is not modified by its presence.

Animals↗
PubMed1997

The testis and the adrenal are (transcriptionally) the same.

We have been studying the transcriptional regulation of the rat P450c17 gene in both adrenocortical and Leydig cells, to assess which DNA sequences are required for its basal and hormonally stimulated transcription. Comparing the transcriptional regulation in both of these cell types enables us to demonstrate whether specific nuclear factors required for transcriptional regulation of the rat P450c17 gene are tissue-specifically expressed, and whether the same cis-acting DNA elements in the gene are required for transcriptional regulation in both of these two different steroidogenic tissues. Using such an approach, we previously demonstrated that the transcriptional regulation of the rat P450scc gene uses different cis-acting DNA sequences in steroidogenic versus neural tissues, and requires the expression of tissue-specific nuclear factors that are unique to neural tissue. However, in studying the transcriptional regulation of the rat P450c17 gene in cultured mouse adrenocortical Y-1 and mouse Leydig MA-10 cells, we have shown that identical DNA sequences necessary for basal and cAMP-stimulated transcriptional regulation in these two cell types, and that identical nuclear factors from Y-1 and from MA-10 cells bind to these sequences. We have identified four transcriptionally active regions within 500 bp of the transcription initiation start site that are important for basal and/or cAMP-stimulated transcriptional regulation of this gene in Y-1 and MA-10 cells. This paper will discuss two of these regions in greater detail. By studying the regulation of the rat P450c17 gene, we have identified two new members of the orphan nuclear receptor gene family and have discovered new alternative mechanisms by which orphan nuclear receptors activate gene transcription in both mouse adrenocortical Y-1 and Leydig MA-10 cells.

Adrenal Glands↗
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↗