Y1 Mouse Adrenocortical Cells Research Articles

THE ROLE OF ERK2/1 IN STEROID PRODUCTION AND StAR PROTEIN EXPRESSION BY Y1 CELLS: STUDIES USING StAR OVER-EXPRESSING TRANSFECTS

In studies using inhibitors of MEK-1, the upstream activator of ERK2/1, we have recently demonstrated that the 42 and 44 kDa extracellular signal-regulated kinases (ERK2/1, respectively) play a major role in regulating cyclic AMP-stimulated StAR gene expression in Y1 mouse adrenocortical cells. We have now extended these studies with the use of a stably-transfected Y1 cell-line over-expressing StAR mRNA under control of CMV promoter control. In these cells, increased StAR expression was correlated with enhanced basal steroidogenesis that was not inhibited by MEK-1 inhibitors (untransfected: 0.7 ± 0.3 pmol pregnenolone/106 cells, transfected: 4.8 ± 1.7 pmol pregnenolone/106 cells), in accordance with ERK2/1 acting to regulate steroid production before transcription of the StAR gene. However, StAR mRNA and protein expression and steroid production by transfected cells were further enhanced in the presence forskolin (FSK, 1 µM) and to a much greater extent than in control cells (untransfected: 4.4 ± 0.2 pmol pregnenolone/106 cells, transfected: 10.4 ± 0.3 pmol pregnenolone/106 cells). Furthermore, MEK-1 inhibitors did not reduce the FSK-induced increase in StAR mRNA and protein expression in transfected cells. These results suggest that activation of adenylate cyclase by FSK can regulate the expression of StAR by both increasing transcription of the endogenous gene and by enhancing stability of existing mRNA. They further suggest that ERK2/1 activation is required for FSK-induced StAR mRNA transcription but is not involved in the regulation of mRNA stability by FSK.

Cfos and cjun antisense oligonucleotides block mitogenesis triggered by fibroblast growth factor-2 and ACTH in mouse Y1 adrenocortical cells.

In G(0)/G(1) cell cycle-arrested mouse Y1 adrenocortical cells, short pulses (30 min to 2 h) of fibroblast growth factor-2 (FGF2) (5 pM to 1 nM) caused induction of cFos protein by 2 h and onset of DNA synthesis stimulation by 8-9 h. FGF2 dose-response curves for cFos induction (percent labeled nuclei with a specific anti-cFos antibody) and DNA synthesis stimulation (bromodeoxyuridine labeling index) were linearly correlated with a correlation coefficient of 0.969. Inhibition of cFos and cJun protein induction with antisense oligodeoxynucleotides (ODNs) to cfos and cjun mRNAs blocked DNA synthesis stimulation by FGF2. Pulses (up to 2 h) of synthetic ACTH(39) (1 pM to 1 nM) and natural porcine corticotropin A (10 pg/ml to 1 microg/ml) also induced cFos protein and DNA synthesis in G(0)/G(1)-arrested Y1 adrenal cells. ACTH dose-response curves for cFos induction and DNA synthesis stimulation were not correlated. But cfos and/or cjun antisense ODNs blocked DNA synthesis stimulation by ACTH. Thus, signals initiated in FGF2 and ACTH receptors appear to converge to the induction of cfos and cjun genes to trigger DNA synthesis stimulation.

Cyclic AMP-induced expression of steroidogenic acute regulatory protein is dependent upon phosphoprotein phosphatase activities.

In addition to the well-documented role of protein kinases in the regulation of steroid production, phosphoprotein phosphatase (PP) activity is required for steroidogenesis. In the present study, we have used the mouse Y1 adrenocortical cell line to identify the site of action of PPs on steroid production by measuring the effects of PP inhibition on the expression of the steroidogenic acute regulatory (StAR) protein and on steroid production. Forskolin-induced activation of cyclic AMP-dependent protein kinase (PKA) enhanced steroidogenesis and this was accompanied by an increased expression of StAR protein. Both steroidogenesis and StAR protein expression were inhibited by two structurally dissimilar inhibitors of PP1 and PP2A activities, okadaic acid and calyculin A. These results suggest that inhibition of PP1 and PP2A inhibits steroid production by preventing the expression of the StAR protein, implicating PP1/2A dephosphorylation reactions as important regulators of stimulus-dependent StAR protein expression, and thus of steroidogenesis.

Three zinc finger nuclear proteins, Sp1, Sp3, and a ZBP-89 homologue, bind to the cyclic adenosine monophosphate-responsive sequence of the bovine adrenodoxin gene and regulate transcription.

Adrenocorticotropin acting through cyclic adenosine monophosphate (cAMP) regulates transcription of the bovine adrenodoxin (Adx) gene in the adrenal cortex. The bovine Adx cAMP-responsive transcription sequence (CRS) has previously been found to contain two consensus GC boxes. By use of nuclear extracts from adrenocortical cells, Sp1 and Sp3 are shown here to bind to CRS. Mutations designed to enhance the identification of additional CRS binding proteins by reducing Sp protein binding showed the presence of an additional DNA-binding protein (Adx factor). Adx factor binding is inhibited by the zinc-chelating agent, 1,10-o-phenanthroline, suggesting it might be a zinc finger protein. By a fractionation/renaturation technique the Adx factor in mouse Y1 adrenocortical cells was found to be in the size range of 106-115 kDa by gel mobility shift assay. On the basis of size, the CRS sequence to which it binds, and its tentative identification as a zinc finger protein, Adx factor has been identified as a Krüppel-like zinc finger protein (a mouse ZBP-89 homologue). Further mutagenesis of CRS demonstrates that it can further be divided into two similar cAMP-responsive elements, and elimination of ZBP-89 binding does not affect cAMP responsiveness of either. Expression of these three nuclear proteins in Drosophila SL2 cells has been used to decipher the role of Adx CRS binding proteins in regulating transcription. Sp1 and Sp3 confer basal transcriptional activities, yet only Sp1 confers cAMP-responsive activity. ZBP-89 represses basal transcriptional activity.

Acth Inhibits a Ras-Dependent Anti-Apoptotic and Mitogenic Pathway in Mouse Y1 Adrenocortical Cells

Mouse Y1 adrenocortical tumor cells harbor amplified and overexpressed c-Ki-ras gene, displaying relatively high constitutive levels of Ras•P. Here we report that Y1 cells also constitutively display high levels of phosphorylated AKT/PKB, that are dependent on Ras•P and PI3K. ACTH rapidly causes dephosphorylation of AKT/PKB in a cAMP/PKA dependent maner. This ACTH inhibition of the anti-apoptic and mitogenic AKT/PKB pathway is likely to be relevant in ACTH growth inhibitory effects in Y-1 adrenocortical cells.

A Role for the p42/44 Isoforms of Mapk in the Regulation of Steroid Secretion from Y1 Mouse Adrenocortical Cells

A Role for the p42/44 Isoforms of Mapk in the Regulation of Steroid Secretion from Y1 Mouse Adrenocortical Cells

Signal Transduction in G0/G1-Arrested Mouse Y1 Adrenocortical Cells Stimulated by Acth and FGF2

In G0/G1 cell cycle arrested mouse Y1 adrenocortical tumor cells ACTH39, a weak mitogen and strong anti-mitogenic agent, blocks FGF2 mitogenic activity at G1 phase, keeping untouched ERK-MAPK activation and c-Fos protein induction. Here we report two anti-mitogenic mechanisms initiated in ACTH receptors and mediated by cAMP/PKA: a) post-transcriptional down regulation of c-Myc protein; b) dephosphorylation of AKT/PKB. In Y-1 cells the activity of the Mad/Max/Myc network of transcription factors seems to be regulated by c-Myc levels. FGF2 induces c-myc gene and stabilizes c-Myc protein by a process dependent on ERK-MAPK (PD98059 sensitive), but not on PI3K (Wortmannin resistant). ACTH39, on the other hand, causes rapid decrease in c-Myc levels induced by FGF2 in wild type Y1 cells, but not in PKA-deficient Y1 clones. The ACTH inhibition of DNA synthesis stimulated by FGF2 is reversed by transient transfection and induction of the MycER chimera (fusion of c-Myc and estrogen-receptor), suggesting that c-Myc down regulation is an efficient anti-mitogenic mechanism activated by ACTH. Y1 cells display high constitutive levels of AKT/PKB, that is dependent on elevated RasGTP. FGF2 up regulates RasGTP, PI3K and AKT/PKB. ACTH antagonizes this mitogenic effect of FGF2, promoting rapid dephosphorylation of AKT/PKB.

Adrenal Tumorigenesis Targeted by the Corticotropin-Regulated Promoter of the Aldo-Keto Reductase AKR1B7 Gene in Transgenic Mice

Studies of ACTH functions in adrenal steroidogenesis have been facilitated by the availability of immortalized mouse adrenocortical Y1 cells. In order to obtain alternative cell lines with a more differentiated zona fasciculata (ZF) phenotype we used targeted tumorigenesis strategy. We have generated transgenic mice expressing the SV40 T antigen under the control of the ACTH-dependent promoter for the AKR1B7/MVDP gene (aldo-keto reductase 1B7/mouse vas deferens protein), which encodes an enzyme responsible for detoxifying isocaproaldehyde, the product of side-chain cleavage of cholesterol generated by steroidogenesis. Our previous data indicated that in the mouse adrenal, AKR1B7 expression was restricted to the ZF and that a 0.5-kb promoter region was able to target specific adrenal expression in transgenic mice. In situ hybridization analyses indicate that AKR1B7 expression during fetal and post-natal periods paralleled the onset of glucocorticoid synthesis and the development of ZF. In transgenic mice. ACTH control and developmental programming of the CAT gene driven by the 0.5-kb promoter followed endogenous gene regulation. Then transgenic mice harboring the 0.5-kb/SV40 T antigen construct were generated and two founders out of three developed adrenal tumors. Cells derived from the tumor of founder 1 (ATC1) were grown in presence of forskolin to maintain ACTH receptor expression and were tested for ACTH responsiveness by immunocychemistry and northern blot analyses. Even after several passages, the ACTH induced AKR1B7 and P450c11β mRNAs accumulations were similar to that observed in mouse primary adrenocortical cell cultures. Our findings suggest that ATC1 cells have conserved essential features of ZF cells. In order to achieve complete characterization of these cells further analyses are currently performed to investigate their steroidogenic activity.

Structure-activity relationship for inhibition of CYP11B1-dependent glucocorticoid synthesis in Y1 cells by aryl methyl sulfones.

The effects of xenobiotics on CYP11B1-dependent corticosterone synthesis (11 beta-hydroxylase) in mouse adrenocortical Y1 cells were studied. 3-Methylsulfonyl-2,2-bis(4-chlorophenyl)-1,1-dichloroethene (MeSO2-DDE) and some methylsulfonyl polychlorinated biphenyls (MeSO2-PCB) inhibited the corticosterone synthesis, whereas PCBs or DDE did not. This indicates a crucial role of the methyl sulfone group for this inhibitory effect. Kinetic analyses of MeSO2-DDE and the two most potent MeSO2-PCBs were conducted using Lineweaver-Burk double-reciprocal plots. The data showed a competitive inhibition of CYP11B1 by the compounds, with apparent inhibitory constants (Ki) of 1.6, 4.6, and 6.7 microM for MeSO2-DDE, 4-MeSO2-2,3,6,4'-tetrachlorobiphenyl, and 4-MeSO2-2,3,6,3',4'-pentachlorobiphenyl, respectively. For comparison, the substrate K(m) was 3.5 microM in the cells, and metyrapone and ketoconazole had apparent Ki-values of 0.8 and 0.04 microM, respectively. In contrast to all previously known inhibitors of CYP11B1, the aryl methyl sulfones are the first examples of CYP11B1 inhibitors not being heterocyclic amines or steroids. The aryl methyl sulfones are widespread environmental pollutants and their inhibition of CYP11B1 constitutes another potential mechanism for endocrine disruption. Their influence on the synthesis of adrenocortical hormones thus merits further interest.

Function of Steroidogenic Factor 1 (SF1) Ligand-Binding Domain in Gene Activation and Interaction with AP1

The nuclear receptor SF1 is an essential mediator in ventromedial hypothalamus–pituitary–gonadal development. As with other nuclear receptors, SF1 possesses a DNA-binding domain composed of two zinc fingers and a ligand-binding domain containing a ligand-dependent activation sequence termed AF2. To dissect the domain function of SF1, we examined various SF1 mutants in mouse adrenocortical Y1 cells and human placental JEG3 cells. Destruction of the AF2 structure removed 73–90% transactivation activity, suggesting that AF2 is indispensable for transactivation. Mutants carrying the DNA-binding domain but lacking the AF2 or the ligand-binding domain blocked the activity of normal SF1. Disrupting the zinc finger diminished the dominant negative effect of mutant. Cotransfection of SF1 with AP1 showed that the two transcription factors cooperated to activate gene expression. Some mutants lost the synergistic action with AP1, while some retained partial activity. These experiments delineate the functional domains of SF1.

Adrenocorticotropic hormone stimulates CYP11B1 gene transcription through a mechanism involving AP-1 factors.

To elucidate the mechanism(s) by which adrenocorticotropic hormone (corticotropin) stimulates transcription of the steroid 11beta-monooxygenase gene (CYP11B1) in adrenocortical cells, the 5'-flanking region of rat CYP11B1 was analyzed using transient transfection and protein-binding assays with mouse adrenocortical Y1 cells. The results indicated that both basal and corticotropin-induced transcriptional activation of CYP11B1 required a common regulatory element containing a binding site for activator protein-1 (AP-1) transcription factors (dimers of the Jun and Fos family proteins) in the 5'-flanking region. Other DNA-binding protein(s) such as transcription factor Ad4BP was not required for either basal or corticotropin-induced transcriptional activation. Corticotropin stimuli were found to induce expression of a subset of the jun and fos family gene products in Y1 cells significantly, while total amounts of AP-1 factors capable of binding to its site in the CYP11B1 promoter did not change greatly. Treatment of rats with corticotropin had similar effects on mRNA levels of the jun and fos family genes in the adrenocortical zona fasciculata cells together with an enhancing effect on the level of CYP11B1 mRNA in the tissue. The effects of corticotropin on mRNA levels of the jun and fos family genes as well as transcription of CYP11B1 in Y1 cells were mimicked by treatment of the cells with dibutyryl cAMP. Furthermore, when components of AP-1 factors were overexpressed by transfecting Y1 cells with their expression vectors, a paired expression of AP-1 components such as c-Jun and c-Fos, which were inducible by corticotropin, transactivated the CYP11B1 promoter more strongly in the absence of corticotropin than other combinations such as JunD and Fra-2 expressed constitutively. These results suggest that corticotropin regulates transcription of the CYP11B1 gene by causing compositional changes in AP-1 transcription factors in the adrenocortical cells via a cAMP-dependent pathway.

The Mouse Adrenocorticotropin Receptor Gene: Cloning and Characterization of Its Promoter and Evidence for a Role for the Orphan Nuclear Receptor Steroidogenic Factor 1

To elucidate the mechanism underlying the tissue-specific expression of the ACTH receptor/MC2 receptor (ACTH-R) in the adrenal cortex, we have cloned the mouse ACTH-R promoter. The analysis of the cDNA 5'-end showed an untranslated region of 321 bp, and the ACTH-R gene was demonstrated to be composed of two exons of 113 and 112 bp lying upstream of the single coding exon. S1 nuclease protection assay showed two major transcription start sites separated by 4 bp; 1.8 kb of the 5'-flanking region inserted in a luciferase reporter vector had cell-specific promoter activity because it was functional only in mouse adrenocortical Y1 cells but not in mouse Leydig TM3 cells or fibroblast L cells. There was no dramatic change in the promoter activity in Y1 cells for all the deletions tested up to -113 bp upstream of the transcription start site. In contrast, expression in TM3 cells was switched on from deletion -908 bp, while remaining undetectable in L cells. Site-directed mutagenesis of a steroidogenic factor 1 (SF1)-like site at position -25 bp resulted in a significant reduction in luciferase expression by the promoter in Y1 cells. Gel shift analysis of this site indicated specific binding of a protein in extracts of Y1 and TM3 cells. Moreover, expression of SF1 in L cells induced promoter activity of the construct p(908). In conclusion, cell-specific expression of the mouse ACTH-R appears to be controlled by at least two factors. One of them, most probably SF1, is responsible for steroidogenic cell-specific expression. The other as yet unknown factor binding between position -1236 bp and -908 bp acts as a strong inhibitory factor in nonadrenal steroidogenic cells, resulting in the adrenal-specific expression of ACTH-R.

The Mouse Adrenocorticotropin Receptor Gene: Cloning and Characterization of Its Promoter and Evidence for a Role for the Orphan Nuclear Receptor Steroidogenic Factor 1

To elucidate the mechanism underlying the tissue-specific expression of the ACTH receptor/MC2 receptor (ACTH-R) in the adrenal cortex, we have cloned the mouse ACTH-R promoter. The analysis of the cDNA 5′-end showed an untranslated region of 321 bp, and the ACTH-R gene was demonstrated to be composed of two exons of 113 and 112 bp lying upstream of the single coding exon. S1 nuclease protection assay showed two major transcription start sites separated by 4 bp; 1.8 kb of the 5′-flanking region inserted in a luciferase reporter vector had cell-specific promoter activity because it was functional only in mouse adrenocortical Y1 cells but not in mouse Leydig TM3 cells or fibroblast L cells. There was no dramatic change in the promoter activity in Y1 cells for all the deletions tested up to− 113 bp upstream of the transcription start site. In contrast, expression in TM3 cells was switched on from deletion −908 bp, while remaining undetectable in L cells. Site-directed mutagenesis of a steroidogenic factor 1 (SF1)-like site at position −25 bp resulted in a significant reduction in luciferase expression by the promoter in Y1 cells. Gel shift analysis of this site indicated specific binding of a protein in extracts of Y1 and TM3 cells. Moreover, expression of SF1 in L cells induced promoter activity of the construct p(908). In conclusion, cell-specific expression of the mouse ACTH-R appears to be controlled by at least two factors. One of them, most probably SF1, is responsible for steroidogenic cell-specific expression. The other as yet unknown factor binding between position −1236 bp and −908 bp acts as a strong inhibitory factor in nonadrenal steroidogenic cells, resulting in the adrenal-specific expression of ACTH-R.

Steroidogenic factor 1 (SF-1) and SP1 are required for regulation of bovine CYP11A gene expression in bovine luteal cells and adrenal Y1 cells.

Cholesterol side-chain cleavage cytochrome P450 (CYP11A; P450scc) gene expression is regulated by gonadotropins via cAMP in the ovary and by ACTH via cAMP in adrenal cortical cells. Previously, we have characterized a response element located at -118 to -101 bp in the 5'-flanking region of the bovine P450scc gene required for cAMP-stimulated transcription in both mouse adrenocortical Y1 cells and bovine ovarian cells in primary culture. It was shown that this region contains a binding site for the transcription factor Sp1. Deletion of this sequence abolished cAMP-stimulated transcription in both Y1 cells and bovine ovarian luteal cells. Another sequence element located at -57 to -32 bp upstream from the transcription initiation site, which is highly conserved in CYP11A of other species, contains the motif TAGCCTTG, similar to the consensus binding site of steroidogenic factor-1, SF-1 (or Ad4-BP), but in the inverted orientation. In the present study, gel shift analysis using nuclear extracts of either Y1 cells or bovine luteal cells demonstrated that the sequence between -57 and -32 bp bound SF-1. A mutation of the SF-1-binding site that abolished binding of the nuclear protein to DNA reduced markedly the basal transcription of the reporter gene as well as the responsiveness to cAMP, when the mutated fragments containing the region from -186 to +12 bp were cloned into a luciferase construct and transfected into mouse adrenal Y1 cells and bovine luteal cells. The role of SF-1 in P450scc transcription was further confirmed by transactivation of the -186/+12Luc construct employing an SF-1 expression vector after transfection into nonsteroidogenic COS-1 cells. In addition, results obtained employing a double mutation of the Sp1- and SF-1-binding sites, and from a construct containing both Sp1 and SF-1 elements upstream of the CYP11A TATA box, indicated that Sp1 and SF-1 function cooperatively in the transactivation of the bovine CYP11A promoter in both bovine luteal cells and Y1 cells. Finally, a mammalian two-hybrid system was employed to demonstrate that Sp1 and SF-1 can associate in vivo. These results establish that basal and cAMP-stimulated activity of the bovine P450scc promoter in both Y1 cells and bovine luteal cells requires the combined action of at least two transcription factors, Sp1 and SF-1.

A cAMP-regulatory sequence (CRS1) of CYP17 is a cellular target for the homeodomain protein Pbx1.

Cytochrome P450c17 encoded by CYP17, whose expression is regulated by peptide hormones via cAMP, is required for cortisol and sex hormone biosynthesis thereby playing a key role in biological processes including sexual differentiation. Utilizing the cAMP-regulatory sequence CRS1 of the bovine CYP17 gene as an affinity ligand, four CRS1-binding proteins have been purified from nuclear extracts of mouse adrenocortical Y1 cells and shown to enhance the in vitro transcription of a reporter gene promoted by CRS1. Microsequencing of these four proteins established two of them to be the homeodomain proteins Pbx1a and Pbx1b, originally discovered by their involvement in the t(1;19) chromosomal translocation in pre-B-cell acute lymphoblastic leukemias. Overexpression of Pbx1 in Y1 cells enhances cAMP-dependent transcription of the CRS1-dependent reporter gene. These results identify the CRS1 of bovine CYP17 as a cellular target for Pbx1 and suggest that one role of this homeodomain protein is in the regulation of steroidogenesis and subsequently sexual development.

Stable transfection of rat angiotensin II receptor subtypes in mouse adrenocortical Y1 cells

A procedure is described for stably transfecting mouse adrenocortical Y1 cells with AT1a and AT1b angiotensin II (AII) receptor subtypes using the lipofectin reagent, DOTAP. The transfected cells are screened by radioreceptor assay using125I-[Sar1,Ile8]AII to select those cells that express high levels of the AII receptors. The establishment of individual transfected Y1 cell lines expressing AT1a and AT1b receptors provides a valuable model system in which to compare the function and signal transduction mechanisms of these two highly homologous AII receptor subtypes.

Isolation and characterization of rat CYP11B genes involved in late steps of mineralo- and glucocorticoid syntheses.

We isolated and characterized four forms of rat CYP11B genes, which were tentatively named CYP11B1, -B2, -B3, and -B4. Genomic Southern analyses indicated that the members of the rat CYP11B gene subfamily were confined to these four genes; among them, CYP11B1 and -B2 encoded steroid 11 beta-hydroxylase and aldosterone synthase, respectively, while CYP11B3 was a gene highly homologous to CYP11B1 without a known expression product. By being devoid of a region spanning two exons conserved in the other three, CYP11B4 was presumably a pseudogene. In the nucleotide sequences, CYP11B1, -B3, and -B4 showed 95-96 and 93-100% identities in the coding and 0.5-kilobase 5'-flanking regions, respectively. However, the homology between the nucleotide sequences of one of the three and CYP11B2 was rather low, about 90 and 50% in the coding and 0.5-kilobase 5'-flanking regions, respectively. As a whole, CYP11B2 rather than CYP11B1, -B3, or -B4 was more homologous to CYP11B genes of other animals such as cow and human. In transient transfection experiments using mouse adrenocortical Y1 cells and chloramphenicol acetyltransferase gene constructs, the 0.5-kilobase 5'-flanking region of CYP11B1 had a 4- and 10-fold higher promoter activity than the corresponding regions of CYP11B2 and -B3, respectively. The possible presence of a suppressive element(s) was noted in the upstream of the 0.5-kilobase region of CYP11B1. Although a variant of cAMP-responsive element, which was present in rat CYP11B2 and all known CYP11B genes of other animals, was modified in rat CYP11B1 and -B3 genes, dibutyryl cAMP stimulated all the promoter activities of the 5'-flanking regions of the rat genes by 3-fold.

The Orphan Nuclear Receptor NGFI-B Regulates Expression of the Gene Encoding Steroid 21-Hydroxylase

As part of its trophic action to maintain the steroidogenic capacity of adrenocortical cells, corticotropin (ACTH) increases the transcription of the cytochrome P-450 steroid hydroxylase genes, including the gene encoding steroid 21-hydroxylase (21-OHase). We previously identified several promoter elements that regulate 21-OHase gene expression in mouse Y1 adrenocortical tumor cells. One of these elements, located at nucleotide -65, closely resembles the recognition sequence of the orphan nuclear receptor NGFI-B, suggesting that NGFI-B regulates this essential steroidogenic enzyme. To explore this possibility, we first used in situ hybridization to demonstrate high levels of NGFI-B transcripts in the adrenal cortex of the adult rat. In cultured mouse Y1 adrenocortical cells, treatment with ACTH, the major regulator of 21-OHase transcription, rapidly increased NGFI-B expression. Gel mobility shift and DNase I footprinting experiments showed that recombinantly expressed NGFI-B interacts specifically with the 21-OHase -65 element and identified one complex formed by Y1 extracts and the 21-OHase -65 element that contains NGFI-B. Expression of NGFI-B significantly augmented the activity of the intact 21-OHase promoter, while mutations of the -65 element that abolish NGFI-B binding markedly diminished NGFI-B-mediated transcriptional activation. Specific mutations of NGFI-B shown previously to impair either DNA binding or transcriptional activation diminished the effect of NGFI-B coexpression on 21-OHase expression. Finally, an oligonucleotide containing the NGFI-B response element conferred ACTH response to a core promoter from the prolactin gene, showing that this element is sufficient for ACTH induction. Collectively, these results identify a cellular promoter element that is regulated by NGFI-B and implicate NGFI-B in the transcriptional induction of 21-OHase by ACTH.

The orphan nuclear receptor NGFI-B regulates expression of the gene encoding steroid 21-hydroxylase.

As part of its trophic action to maintain the steroidogenic capacity of adrenocortical cells, corticotropin (ACTH) increases the transcription of the cytochrome P-450 steroid hydroxylase genes, including the gene encoding steroid 21-hydroxylase (21-OHase). We previously identified several promoter elements that regulate 21-OHase gene expression in mouse Y1 adrenocortical tumor cells. One of these elements, located at nucleotide -65, closely resembles the recognition sequence of the orphan nuclear receptor NGFI-B, suggesting that NGFI-B regulates this essential steroidogenic enzyme. To explore this possibility, we first used in situ hybridization to demonstrate high levels of NGFI-B transcripts in the adrenal cortex of the adult rat. In cultured mouse Y1 adrenocortical cells, treatment with ACTH, the major regulator of 21-OHase transcription, rapidly increased NGFI-B expression. Gel mobility shift and DNase I footprinting experiments showed that recombinantly expressed NGFI-B interacts specifically with the 21-OHase -65 element and identified one complex formed by Y1 extracts and the 21-OHase -65 element that contains NGFI-B. Expression of NGFI-B significantly augmented the activity of the intact 21-OHase promoter, while mutations of the -65 element that abolish NGFI-B binding markedly diminished NGFI-B-mediated transcriptional activation. Specific mutations of NGFI-B shown previously to impair either DNA binding or transcriptional activation diminished the effect of NGFI-B coexpression on 21-OHase expression. Finally, an oligonucleotide containing the NGFI-B response element conferred ACTH response to a core promoter from the prolactin gene, showing that this element is sufficient for ACTH induction. Collectively, these results identify a cellular promoter element that is regulated by NGFI-B and implicate NGFI-B in the transcriptional induction of 21-OHase by ACTH.

Elevated levels of protein kinase C in Y1 cells which express apolipoprotein E decrease basal steroidogenesis by inhibiting expression of P450-cholesterol side chain cleavage mRNA.

We have previously reported that steroidogenesis is dramatically reduced in mouse Y1 adrenocortical cells which express the human apolipoprotein E gene (Y1-E cells). This suppression results in part from inhibition of cAMP-mediated events. In this report we have examined the expression of protein kinase C (PKC) in the Y1-E cell lines. Total cellular PKC activity in vitro is increased 3-5-fold in the Y1-E cell lines. PKC activity in the particulate and cytosolic fractions is increased to the same relative extent. Increased PKC activity reflects increased levels of PKC mRNA, as determined by Northern blot analysis, and PKC protein, as determined by immunoblot analysis. Increased expression of PKC in the Y1-E cell lines is accompanied by a 2-3-fold increase in diacylglycerol, an in vivo activator of PKC. To determine the contribution of elevated PKC expression to the Y1-E cell phenotype, we utilized the PKC inhibitors, staurosporine and calphostin C. Upon treatment with staurosporine or calphostin C, expression of P450-cholesterol side chain cleavage mRNA is increased severalfold to a level equal to, or greater than, basal expression in the Y1-neo control cell line. Treatment with calphostin C also results in recovery of steroidogenesis in the Y1-E cells to a level comparable to the basal level observed in the Y1-neo control cell line. These results indicate that increased expression of PKC in the Y1-E cell lines decreases basal steroidogenesis by suppressing P450-cholesterol side chain cleavage mRNA expression. Inhibition of PKC, however, does not reverse the block in cAMP-stimulated steroidogenesis in Y1-E cells, suggesting that the pleiotropic effects of apoE expression are not mediated entirely through altered PKC expression.