Y1 Mouse Adrenocortical Tumor Cells Research Articles

Impaired steroidogenic factor 1 (NR5A1) activity in mutant Y1 mouse adrenocortical tumor cells.

Mutants isolated from the Y1 mouse adrenocortical tumor cell line (clones 10r-9 and 10r-6) are resistant to ACTH because they fail to express the melanocortin-2 receptor (MC2R). In this study, we show that a luciferase reporter plasmid driven by 1,800 bp of the proximal promoter region of the MC2R was expressed poorly in the mutant cells compared with parent Y1 cells. The differential expression of the MC2R in parent and mutant cells resulted from impaired activity of the orphan nuclear receptor NR5A1 (SF1) on the promoter as determined by 5'-deletion analysis. Furthermore, the activity of an SF1 expression plasmid on an SF1-dependent reporter plasmid was compromised in mutant clones. The site-specific DNA binding properties of SF1 from parent and mutant cells did not differ as determined in electrophoretic mobility shift assays, and the addition of the activation domain of VP16 to the amino terminus of SF1 restored the transcriptional activity of the protein. In addition, the levels of SF1 and other cofactors including WT1, CBP/p300, and steroid receptor coactivator 1 did not differ appreciably between parent and mutant cells. Taken together, these results suggest that ACTH resistance in the mutant clones resulted from a defect that affected the activation properties of SF1 rather than its DNA binding activity. Consistent with the observed impairment in SF1 function, other SF1-dependent genes, including Cyp11b1 and steroidogenic acute regulatory protein (StAR), were poorly expressed and global steroidogenesis, as evidenced by the metabolism of 22(R)-hydroxycholesterol to steroid products, was impaired. Interestingly, MC2R, Cyp11a, Cyp11b1, and StAR transcripts were not affected to the same degree, suggesting that each of these genes may have a different absolute requirement for SF1. These mutants thus provide an experimental paradigm to identify factors that influence SF1 function and to evaluate the relative importance of SF1 in the expression of genes essential for adrenal steroidogenesis.

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.

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.

Role of ERK/MAP Kinase in Mitogenic Interaction Between Acth and FGF2 in Mouse Y1 Adrenocortical Tumor Cells

In G0/G1 cell cycle-arrested Y1 adrenocortical cells FGF2 is a strong mitogen, whereas ACTH39 can be a weak mitogen or a strong anti-mitogenic agent Phosphorylated ERK1/2-MAP kinases are undetectable by Western and immunocitochemistry assay in G0/G1-arrested Y1 adrenal cells. Cell entry into S phase linearly correlates with migration of phosphorylated ERK to nucleus. FGF2 rapid and strongly triggers transient phosphorylation of ERK1/2, whereas ACTH39 is a poor ERK1/2 activator. But, the MEK1 inhibitor, PD98059 (50μM), inhibits cFos and cyclin D1 induction and DNA synthesis stimulation by both ACTH39 and FGF2, suggesting that ERK1/2 activation mediates the strong and the weak mitogenic effect of, respectively, FGF2 and ACTH39 In addition, ACTH39 antagonizes the FGF2 mitogenic effect keeping untouched ERK1/2 activation, c-Fos and cyclin D1 induction.

A role for guanyl nucleotide-binding regulatory protein beta- and gamma-subunits in the expression of the adrenocorticotropin receptor.

Mutant Y1 mouse adrenocortical tumor cells, isolated on the basis of their resistance to the growth-inhibitory effects of forskolin, arise from single mutational events. These mutants present complex phenotypes in which the activity of Gbeta/gamma is impaired, ACTH receptor gene expression is markedly diminished, and ACTH-responsive adenylyl cyclase activity is lost. In this study, we have tested the hypothesis that the impairment in Gbeta/gamma activity is responsible for the loss of ACTH receptor gene expression and ACTH-responsive adenylyl cyclase activity. Transfection of one of the mutant clones with expression vectors encoding either Gbeta1 or Gbeta2 together with Ggamma2 increased ACTH receptor expression and restored ACTH-responsive adenylyl cyclase activity. Interestingly, either Gbeta2 or Ggamma2 alone was effective. These results thus support the hypothesis that the impairment in Gbeta/gamma activity is responsible for the loss of ACTH receptor expression. A luciferase reporter plasmid driven by the proximal promoter region of the mouse ACTH receptor gene was expressed poorly in the mutants compared with parental Y1 cells, suggesting that the Gbeta/gamma defect compromised transcriptional activity at the proximal promoter region of the ACTH receptor gene.

Altered G protein activity in a desensitization-resistant mutant of the Y1 adrenocortical tumor cell line.

Mutant isolates [designated desensitization resistant (DR)] from the Y1 mouse adrenocortical tumor cell line resist agonist-induced desensitization of adenylyl cyclase by preventing the uncoupling of receptors from their guanyl nucleotide-binding regulatory G proteins. In this study, we tested the hypothesis that an underlying G protein defect is associated with the DR phenotype. We found that the G protein reagent guanyl-5'-yl imidodiphosphate [Gpp(NH)p] shifted beta2-adrenergic receptors from a high affinity state to a low affinity state 4-fold more effectively in mutant DR cells than in parent Y1 cells. In the DR mutant, Gpp(NH)p was able to shift receptors to a low affinity state in the absence of NaCl, whereas the effect of Gpp(NH)p in parent Y1 cells was dependent upon the presence of NaCl. Moreover, these differences in sensitivity to Gpp(NH)p and NaCl were transferred to Gs alpha-deficient S49(CYC-) lymphoma cell membranes in G protein reconstitution assays. These observations suggested that the DR mutation was associated with altered activity of the stimulatory G protein, Gs. Cloning and sequence analysis demonstrated that Gs alpha transcripts in the DR mutant were normal, suggesting that another factor involved in guanyl nucleotide exchange is responsible for the altered G protein activity in DR mutant cells.

Unmasking a Growth-promoting Effect of the Adrenocorticotropic Hormone in Y1 Mouse Adrenocortical Tumor Cells

The adrenocorticotropic hormone (ACTH) inhibits the growth of Y1 mouse adrenocortical tumor cells as well as normal adrenocortical cells in culture but stimulates adrenocortical cell growth in vivo. In this study, we investigated this paradoxical effect of ACTH on cell proliferation in Y1 adrenal cells and have unmasked a growth-promoting effect of the hormone. Y1 cells were arrested in the G1 phase of the cell cycle by serum starvation and monitored for progression through S phase by measuring [3H]thymidine incorporation into DNA and by measuring the number of nuclei labeled with bromodeoxyuridine. Y1 cells were stimulated to progress through S phase and to divide after a brief pulse of ACTH (up to 2 h). This effect of ACTH appeared to be cAMP independent, since ACTH also induced cell cycle progression in Kin-8, a Y1 mutant with defective cAMP-dependent protein kinase activity. The growth-promoting effect of ACTH in Y1 was preceded by the rapid activation of p44 and p42 mitogen-activated protein kinases and by the accumulation of c-FOS protein. In contrast, continuous treatment with ACTH (14 h) inhibited cell cycle progression in Y1 cells by a cAMP-dependent pathway. The inhibitory effect of ACTH mapped to the midpoint of G1. Together, the results demonstrate a dual effect of ACTH on cell cycle progress, a cAMP-independent growth-promoting effect early in G1 possibly mediated by mitogen-activated protein kinase and c-FOS, and a cAMP-dependent inhibitory effect at mid-G1. It is suggested that the growth-inhibitory effect of ACTH at mid-G1 represents an ACTH-regulated check point that limits cell cycle progression.

Molecular strategies for the dominant inhibition of protein kinase C.

The regulatory (R) domain of PKC alpha fused to glutathione-S-transferase (GST-R alpha) competitively inhibited PKC activity associated with extracts of Y1 mouse adrenocortical tumor cells and the activities of several specific PKC isozymes. GST-R alpha did not inhibit the activities of cAMP-dependent protein kinase, cGMP-dependent protein kinase or calmodulin-dependent myosin light chain kinase. GST-R alpha inhibited PKC activities 20 times more potently than did a synthetic peptide corresponding to the pseudosubstrate sequence of PKC alpha. In intact yeast cells, the R domain prevented PKC beta-1-induced inhibition of growth and cytokinesis. These results indicate that the R domain of PKC alpha acts as a specific, dominant inhibitor of PKC activity, and suggest that the PKC alpha R domain may provide a useful genetic tool to assess the roles of PKC in various signal transduction processes.

Amplification of the Transketolase Gene in Desensitization-resistant Mutant Y1 Mouse Adrenocortical Tumor Cells

As shown previously, mutants of the Y1 mouse adrenocortical tumor cell line that resist agonist-induced desensitization of adenylyl cyclase have elevated levels of a 68-kDa protein (designated p68), suggesting a possible relationship between p68 and the regulation of adenylyl cyclase activity. In the present study, cDNA cloning and sequencing were used to identify p68 as mouse transketolase. Cells overexpressing p68 exhibited a 17.4-fold increase in transketolase enzymatic activity relative to parental Y1 cells and a 28-fold amplification of the transketolase gene as determined by Southern blot hybridization analysis. Using fluorescent in situ hybridization analysis, the transketolase gene was mapped to mouse chromosome 16B1 and to human chromosome 3p21.2. Transketolase gene amplification was associated with telomeric fusion of the chromosome 16 pair together with the appearance of multiple copies of the transketolase gene throughout a different chromosome. The relationship between overexpression of transketolase and desensitization resistance was evaluated in somatic cell hybrids formed between a desensitization-resistant adrenal cell line and a desensitization-sensitive rat glial cell line. In these hybrids, transketolase overexpression behaved dominantly, whereas desensitization resistance behaved recessively. These results dissociate the desensitization resistance phenotype from overexpression of transketolase and suggest that desensitization resistance may have resulted from disruption of an essential regulatory gene in conjunction with the amplification event.

Adrenocorticotropin-resistant mutants of the Y1 adrenal cell line fail to express the adrenocorticotropin receptor.

This report examines the basis for adrenocorticotropin (ACTH) resistance in two mutant clones (Y6 and OS3) derived from the ACTH-responsive Y1 mouse adrenocortical tumor cell line. These two mutants were originally characterized by their failure to respond to ACTH with increased adenylyl cyclase activity and as a consequence were resistant to the steroidogenic effects of the hormone. We now demonstrate that ACTH resistance in the Y6 and OS3 mutants results from the failure to express the gene encoding the ACTH receptor. Whereas parental Y1 cells express ACTH receptor transcripts at low levels and are stimulated by ACTH or 8-bromo-cAMP to increase the accumulation of ACTH receptor transcripts approximately twofold, the Y6 and OS3 mutants do not express receptor transcripts either in the presence or absence of 8-bromo-cAMP. The gene encoding the ACTH receptor appears to be present in the Y6 and OS3 mutants, as determined by Southern blot hybridization analysis. Moreover, in the Y6 mutant the ACTH receptor gene appears to be silenced by a modification that is reversed following the growth of the cells as tumors in mice. Clonal isolates of Y6 cells grown as tumors recover the ability to express ACTH receptor transcripts at low but detectable levels and acquire the ability to respond to ACTH with increased adenylyl cyclase activity. Finally, Y6 and OS3 cells transformed with a gene encoding the mouse beta 2-adrenergic receptor respond to the beta-adrenergic agonist, isoproterenol, in a manner that is indistinguishable from the similarly transformed parent Y1 cell line. These latter results demonstrate the functional integrity of the adenylyl cyclase system in the ACTH-resistant mutants and indicate that the failure to express ACTH receptor transcripts limits the responsiveness of these clones.

ACTH-receptor deficient mutants of the Y1 mouse adrenocortical tumor cell line.

Two mutant clones (Y6 and OS3) derived from the ACTH-responsive Y1 mouse adrenocortical tumor cell line fail to respond to ACTH with increased adenylyl cyclase activity and, as a consequence, are resistant to the steroidogenic effects of the hormone. As determined from Northern blot and RNase protection assays, ACTH resistance in these mutants results from the failure to accumulate ACTH receptor transcripts. The ACTH receptor gene appears to be present in these mutants as determined by Southern blot hybridization analysis and can be activated following the growth of the mutant cells as tumors in mice, suggesting that the ACTH receptor gene is modified in a reversible manner. When mutant cells are transformed with a gene encoding the mouse beta 2-adrenergic receptor they respond to beta-adrenergic agonists with increased adenylyl cyclase activity in a manner that is indistinguishable from a similarly transformed parent Y1 cell line. These results suggest that the adenylyl cyclase system in the mutants is otherwise intact and that the failure to express ACTH receptor transcripts limits the responsiveness of these clones to the hormone.

Receptor-G-protein coupling in desensitization-resistant mutant adrenal cells.

Mutant isolates from the Y1 mouse adrenocortical tumor cell line (designated Y1DR) were previously shown to resist homologous desensitization of adenylyl cyclase via endogenous ACTH receptors or transfected beta 2-adrenergic receptors. To further localize the site of the DR mutation, we examined the agonist-binding properties of the transfected beta 2-adrenergic receptor in parental Y1 cells and the DR mutant before and after desensitization with the beta-adrenergic agonist isoproterenol. Binding studies were carried out using [125I]iodocyanopindolol as the labeled ligand and isoproterenol as the competing agonist. We found that the DR mutant has a greater number of high affinity receptors in the basal state than does the Y1 parent (70-80% vs. < or = 50%) and that treatment of membranes from parent or mutant cells with guanyl-5'-yl imidodiphosphate and NaCl converts all of the high affinity sites to low affinity sites. After desensitization with isoproterenol, only low affinity binding sites are detected in parental Y1 cells, whereas the DR mutants retain an appreciable number of high affinity receptors. These results indicate that the DR mutant may resist desensitization by affecting agonist-induced uncoupling of receptors from their guanyl nucleotide-binding regulatory G-proteins.

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.

3',5'-cyclic adenosine monophosphate-dependent transcription of the CYP11A (cholesterol side chain cleavage cytochrome P450) gene involves a DNA response element containing a putative binding site for transcription factor Sp1.

The product of the CYP11A gene, cholesterol side chain cleavage cytochrome P450, catalyzes the initial step of steroidogenesis. A major mechanism whereby steroid hydroxylase gene transcription is regulated in the adrenal cortex requires the pituitary peptide hormone, ACTH, which acts via cAMP. We have previously identified a transcriptional enhancer in the 5'-flanking sequence [-183 to -83 base pairs (bp)] of the bovine CYP11A gene, which activates transcription of a beta-globin promoter/reporter gene in transiently transfected mouse Y1 adrenocortical tumor cells in response to the activator of adenylate cyclase, forskolin. Further deletion analysis has located the minimal cAMP-responsive sequence (CRS) to -118 to -100 bp. Analysis of DNA-protein interactions using nuclear extracts from Y1 cells revealed two protein binding sites, which were shown by competition analysis to be closely related to the two protein binding sites identified previously in the CRS of the human CYP21 gene. Namely, within the cAMP responsive fragment -118 to -100 bp, a sequence with a high degree of similarity to the consensus binding sequence for the ubiquitous transcription factor Sp1 is present, and binding of protein to this site was abolished by competition with excess GC box oligonucleotide. The second partially overlapping site is located 3' of the putative Sp1-binding site and binds to a protein identical or closely related to a putative adrenal-specific protein. Whereas the adrenal-specific protein binding site of the CYP21 CRS was previously shown to be sufficient to confer cAMP-responsive activation of transcription, the homologous site within the CYP11A CRS appears to have an attenuating effect on transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Adenylyl Cyclase Activity by β-Adrenergic Agonists in a Desensitization-Resistant Mutant Cell Line

Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1DR mutant were transfected with a gene encoding the mouse beta 2-adrenergic receptor, and transfectants isolated from both Y1DS and Y1DR cells were shown to express beta 2-adrenergic receptors. These transfectants responded to the beta-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the beta 2-adrenergic receptor. Treatment of intact Y1DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the beta-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTH-induced desensitization of adenylyl cyclase. As determined using the hydrophilic beta-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1DS and Y1DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants.

Decreased Levels of Guanyl Nucleotide-Binding Regulatory Protein α-Subunits in Y1 Adrenocortical Tumor Cell Mutants Resistant to Forskolin

Forskolin-resistant mutants arise from Y1 mouse adrenocortical tumor cells with a frequency indicative of a mutational event at a single genetic locus and exhibit adenylyl cyclases that are resistant to activation by forskolin, corticotropin, and guanyl-5'-yl-imidodiphosphate. This study examined the levels of guanyl nucleotide-binding regulatory protein subunits (G) in plasma membranes from the forskolin-resistant mutants by Western blot immunoanalysis. In plasma membranes prepared from parental Y1 cells and from four forskolin-resistant mutants, 10r-2, 10r-3, 10r-6, and 10r-9, the levels of the alpha-subunits of Gs and Gi-2 were reduced by 70-80% relative to the levels in parental Y1 cells. The levels of the beta 36-subunit were much less affected, and the levels of the alpha i-3 and beta 35-subunits varied independently of the forskolin-resistant phenotype. As determined by slot blot hybridization analyses, the levels of Gs alpha and Gi alpha RNA in the forskolin-resistant mutants were equivalent to those in the Y1 parent. Therefore, the decreased levels of Gs alpha and Gi alpha-2 subunits observed in the forskolin-resistant mutants did not result from decreased expression of the genes encoding these proteins. Our observations suggest that the forskolin-resistant phenotype of Y1 mutants resulted from single mutations that affected the processing of specific G alpha subunits or their incorporation into the plasma membrane.

CisModification of the Steroid 21-Hydroxylase Gene Prevents Its Expression in the Y1 Mouse Adrenocortical Tumor Cell Line

The Y1 mouse adrenocortical tumor cell line retains the ability to synthesize and secrete steroids, but does not express steroid 21-hydroxylase (C21) and, therefore, does not produce 21-hydroxylated steroids. In this investigation the mechanisms underlying the loss of C21 activity in the Y1 cell line were explored. A 9-kilobase BglII fragment containing the C21 gene was cloned from the Y1 genome. This genomic clone directed the synthesis of C21 transcripts and 21-hydroxylated steroid products when transfected back into the Y1 cell line. As determined by restriction endonuclease digestions with MspI and HpaII, enzymes that distinguish between unmethylated and methylated CCGG sites, the endogenous C21 gene was extensively methylated in Y1 adrenal cells and in cells from other mouse tissues that do not normally express this gene. In contrast, the C21 gene was hypomethylated in primary cultures of mouse adrenal cells which normally synthesize large amounts of C21. The cloned C21 gene transfected into Y1 cells initially was unmethylated, but became extensively methylated with prolonged culture of the cells; prolonged culture of these transfectants also resulted in a loss of C21 expression. Loss of C21 expression in Y1 transfectants, however, temporally preceded the extensive methylation of the transfected C21 gene. Furthermore, treatment of Y1 cells with 5-azacytidine caused a demethylation of the endogenous C21 gene, but did not result in the recovery of C21 expression. These results indicate that Y1 cells contain a functional C21 gene that has been silenced by a reversible cis-modification event.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the promoter region of the gene encoding mouse cholesterol side-chain cleavage enzyme.

The cholesterol side-chain cleavage enzyme (SCC) catalyzes the initial and rate-limiting step in the synthesis of steroid hormones. The mouse gene encoding SCC was cloned and the nucleotide sequence of its 5'-flanking region determined. This sequence includes an AP-1 motif at -319 and two motifs, AGGTCA at -70 and AGCCTTG at -40, that match elements proposed to be important in the expression of steroid 21-hydroxylase. When transfected into mouse Y1 adrenocortical tumor cells, 1.5 kilobase pairs of 5'-flanking region of the SCC gene directed high levels of expression of a growth hormone reporter gene; treatment of the transfected Y1 cells with 8-bromo-cAMP increased this expression by 5-fold. In contrast, transfected mouse MA-10 Leydig cells showed appreciably lower expression, suggesting that SCC expression in Leydig cells requires additional elements not contained in the 5'-flanking region of the SCC gene used in these experiments. Deletion experiments showed that 424 base pairs of 5'-flanking sequences were sufficient for regulated expression in Y1 cells and mapped two regulatory regions: one from -424 to -327 and a second from -219 to -77. DNase I footprinting and gel mobility shift analyses of these 424 base pairs defined several interactions between nuclear proteins and the SCC promoter, including footprints centered over the AP-1 motif, over a sequence at -120, and over the sequences (-70 and -40) that resemble 21-hydroxylase promoter elements. Finally, site-selected mutagenesis of the potential elements at -40, -70, or -120 decreased SCC promoter activity in transfected Y1 adrenocortical cells, thus establishing their importance in SCC expression.

Identification and Characterization of Two Upstream Elements that Regulate Adrenocortical Expression of Steroid 11β-Hydroxylase

We have studied the mechanisms that regulate the expression of the mouse gene encoding steroid 11 beta-hydroxylase (11 beta-OHase), a steroidogenic cytochrome P450 enzyme that is expressed only in the adrenal cortex. DNase I footprinting and gel-mobility shift analyses revealed potential regulatory elements at -370 and -310 in the 11 beta-OHase promoter region. To determine the contributions of these elements to expression, we altered their sequences by site-selected mutagenesis and studied promoter activity after transfection into Y1 mouse adrenocortical tumor cells. Mutation of either element markedly decreased basal promoter activity but did not affect the response to treatment with 8-bromo cAMP. These experiments thus document the functional roles of these elements, within the context of the intact promoter, in constitutive expression of 11 beta-OHase. Moreover, addition of either of these elements to p-40GH, a 5'-deletion plasmid containing 11 beta-OHase sequences from -40 to +8 upstream of a growth hormone reporter gene, significantly increased promoter activity but did not confer cAMP responsiveness. Finally, increased expression was seen after transfection of Y1 derivatives deficient in cAMP-dependent protein kinase, indicating that neither element required cAMP-dependent protein kinase activity. These studies thus define two regulatory elements that play important roles in 11 beta-OHase expression.