Y1 Adrenal Cells Research Articles

Protein kinase C is a tonic negative regulator of steroidogenesis and steroid hydroxylase gene expression in Y1 adrenal cells and functions independently of protein kinase A

Protein kinase C is a tonic negative regulator of steroidogenesis and steroid hydroxylase gene expression in Y1 adrenal cells and functions independently of protein kinase A

Genomic elements involved in transcriptional regulation of the rabbit surfactant protein-A gene.

Expression of the surfactant protein-A (SP-A) gene is lung specific and is developmentally and hormonally regulated in fetal lung tissue. Cyclic AMP analogs and glucocorticoids stimulate transcriptional activity of the SP-A gene in fetal rabbit lung tissue in culture; an additive effect is observed when the agents are added in combination. To analyze the genomic regions that regulate SP-A promoter activity, fusion genes comprised of -1766, -991, -378, and -47 basepairs (bp) of DNA flanking the 5'-end of the SP-A gene, the transcription initiation site, and 20 bp of exon I linked to the human GH (hGH) structural gene were subcloned into a replication-defective human adenovirus vector and transfected into differentiated rat type II cells in primary culture. SP-A promoter activity was analyzed by RIA of hGH protein in the culture medium. In type II cells transfected with SP-A-1766:hGH and SP-A-991:hGH fusion genes, hGH production was induced 30- to 40-fold by (Bu)2AMP (Bt2cAMP; 1 mM). When type II cells were transfected with the SP-A-378:hGH fusion gene, basal levels of expression were reduced by more than 50%; however, Bt2cAMP caused an 11-fold increase in hGH production. In type II cells transfected with the SP-A-47:hGH fusion gene, basal levels of hGH production were essentially undetectable, and no stimulatory effect of Bt2cAMP was apparent. Cyclic AMP stimulation of expression of the SP-A-1766:hGH, SP-A-991:hGH, and SP-A-378:hGH fusion genes was limited to type II pneumonocytes in primary culture and was absent in two lung adenocarcinoma cell lines (NCl-H358 and A549), which do not express SP-A, and in cAMP-responsive adrenal Y1 cells. Mutations of a putative cAMP-responsive element (TGACCTCA) at -261 bp revealed its functional importance in mediating cAMP regulation of SP-A gene expression. Unexpectedly, dexamethasone (Dex; 10(-7) M) antagonized the stimulatory effect of Bt2cAMP on expression of SP-A:hGH fusion genes containing from -378 to -1766 bp of 5'-flanking DNA as well as that of a fusion gene construct containing -991 bp of 5'-flanking DNA, the first exon, the first intron, and 20 bp of the second exon (SP-A-991+670:hGH). The inhibitory effect of Dex was dose dependent, with half-maximal inhibition occurring at a Dex concentration of 8 x 10(-10) M. The inhibitory effect of Dex was prevented by the glucocorticoid receptor antagonist RU486.(ABSTRACT TRUNCATED AT 400 WORDS)

Diminished Functional Activity of Newly Synthesized Muscarinic Acetylcholine Receptors in Stably Transfected Y1 Adrenal Cells

We have examined the functional responsiveness of newly synthesized m2 muscarinic acetylcholine receptors in stably transfected Y1 adrenal cells. After inactivation of preexisting receptors with the covalent alkylating antagonist propylbenzilylcholine mustard, the number of cell surface receptors returned to control values over a 3-h period. After a 3-h recovery, the cells exhibited diminished sensitivity for muscarinic receptor-mediated inhibition of adenylyl cyclase activity, with much higher concentrations of agonist being required to elicit a response. The functional sensitivity returned to control values over a 12-18-h period. The decreased functional activity was not due to a decreased affinity of the newly synthesized receptors for agonist or to a decrease in the levels of inhibitory G proteins in the cells. The results suggest that muscarinic receptors may be synthesized in a form with diminished functional activity. The ability to study the maturation of receptor function in a transfected cell system should allow a combination of biochemical and molecular genetic approaches to analyze the synthesis and functional responsiveness of muscarinic receptors.

Involvement of the Golgi region in the intracellular trafficking of cholera toxin.

The intracellular pathway following receptor-mediated endocytosis of cholera toxin was studied using brefeldin A (BFA), which inhibited protein secretion and induced dramatic morphological changes in the Golgi region. In both mouse Y1 adrenal cells and CHO cells, BFA at 1 micrograms/ml caused a 80-90% inhibition of the cholera toxin (CT)-induced elevation of intracellular cAMP. The inhibition of the cytotoxicity of CT by BFA was also observed in a rounding assay of Y1 adrenal cells. The inhibition of CT cytotoxicity by BFA was dose dependent, with the ID50 value similar to the LD50 of BFA in Y1 adrenal cells. Binding and internalization of [125I]-labeled cholera toxin in Y1 adrenal cells was not affected by BFA. Unlike the BFA-sensitive cell lines such as Y1 adrenal and CHO cells, BFA at 1 micrograms/ml did not inhibit the cytotoxicity of CT in PtK1 cells, of which the Golgi structure was BFA-resistant. These results strongly suggest that a BFA-sensitive Golgi is required for the protection of CT cytotoxicity by BFA. In contrast, elevation of the intracellular cAMP by forskolin, which acts directly on the plasma membrane adenylate cyclase, was not affected by BFA. These observations indicate that the intoxication of target cells by CT requires an intact Golgi region for its intracellular trafficking and/or processing. In this respect, CT shares a common intracellular pathway with ricin, Pseudomonas toxin, and modeccin, even though their structures and modes of action are very different.

The C gamma subunit is a unique isozyme of the cAMP-dependent protein kinase.

There are at least three isozymes (C alpha, C beta, and C gamma) of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase (PKA) (Beebe, S., Oyen, O., Sandberg, M., Froysa, A., Hansson, V., and Jahnsen, T. (1990) Mol. Endocrinol. 4, 465-475). To compare the C gamma and C alpha isozymes, the respective cDNAs were expressed in permanently transformed Kin-8 PKA-deficient Y1 adrenal cells using the mouse metallothionein promoter. The recombinant C subunits were characterized as immunoreactive, zinc-inducible, cAMP-dependent kinase activities. In contrast to C alpha, histone was a better substrate than Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) for C gamma. Furthermore, C gamma histone kinase activity was not inhibited by the protein kinase inhibitor peptide (5-24 amide), which has been widely used as a PKA-specific inhibitor. The major C gamma peak (type I) eluted from DEAE-Sepharose at a higher NaCl concentration (120 mM) than the C alpha type I eluted (70 mM). C gamma and C alpha type II eluted between 220 and 240 mM NaCl. C gamma required higher concentrations of cAMP than C alpha did for dissociation from the mutant type I holoenzyme. These differences provided a basis for the separation of the mutant RI-associated isozymes on DEAE-Sepharose. Both C alpha (41-42 kDa) and C gamma (39-40 kDa) were identified by a C subunit antibody after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. Zinc induced the PKA-mediated rounding phenotype in C gamma and C alpha clones, thereby restoring the cells to the parent Y1 adrenal cell phenotype. Collectively, these data indicate that C gamma is an active PKA C subunit but suggest that C gamma and C alpha have different protein and peptide recognition determinants.

Identification of positive and negative placenta-specific basal elements and a cyclic adenosine 3',5'-monophosphate response element in the human gene for P450scc.

The chronic regulation of steroiodgenesis is mediated principally by transcriptional regulation of the genes encoding the various steroidogenic enzymes. The cholesterol side-chain cleavage enzyme, P450scc, is rate limiting and hormonally regulated in a tissue-specific fashion. Human placental steroidogenesis is regulated by LH and hCG through increased intracellular cAMP, and forskolin and 8-bromo-cAMP increase the abundance of human P450scc mRNA in human JEG-3 choriocarcinoma cells. We transfected JEG-3 cells with 24 promoter/reporter constructions to examine the tissue-specific and hormonally induced transcription of the human P450scc gene in these cells. A reporter construction containing only bases -79 to +49 of the human P450scc gene was expressed in JEG-3 cells. This basal expression was increased by four elements, especially by a powerful element between -152 to -142. Adding DNA sequences to -177 suppressed the basal expression seen with the -152 construction, indicating that a repressor element lies between -177 and -152. Thus, basal expression of the human P450scc gene in JEG-3 cells is mediated by the interplay of several separate cis-acting DNA elements. Forskolin induction was conferred by sequences between -108 and -89. The mechanism for cAMP induction appears to be direct, as this induction is rapid and is not blocked by inhibiting protein synthesis with cycloheximide. Gel mobility shift experiments identified six specific DNA-protein complexes. Five of these complexes correlate closely with the basal transcription activities identified by the reporter assays. The powerful basal element, the repressor element, and the cAMP element differ from those identified by similar experiments in mouse adrenal Y1 cells, suggesting that the human P450scc gene is regulated by the tissue-specific use of different regulatory elements.

Heterologous desensitization of the human dopamine D1 receptor in Y1 adrenal cells and in a desensitization-resistant Y1 mutant

Heterologous desensitization of the human dopamine D1 receptor in Y1 adrenal cells and in a desensitization-resistant Y1 mutant

Reduced mRNA levels for the multidrug-resistance genes in cAMP-dependent protein kinase mutant cell lines.

We have previously shown that in Chinese hamster ovary (CHO) cells, a mutant cell line with a defective regulatory subunit (RI) for the cAMP-dependent protein kinase (Abraham et al: Mol. Cell. Biol., 7:3098-3106, 1987), and a transfectant cell line expressing the same mutant kinase, showed increased sensitivity to a number of drugs that are known to be substrates for the multidrug transporter (P-glycoprotein). In the current study we have investigated the mechanism by which cAMP-dependent protein kinase controls drug resistance. We report here that the sensitivity of the kinase defective CHO cell lines to multiple drugs results from decreased RNA levels for the multidrug-resistance gene. Similar results were obtained with mouse Y1 adrenal cells. Wild-type Y1 cells had high levels of P-glycoprotein due to expression of both the mdr1b and mdr2 genes, whereas the cAMP-dependent protein kinase mutant Kin 8 cells had decreased RNA levels for these genes. A Kin 8 transfectant with restored cAMP-dependent protein kinase activity recovered mdr expression, indicating a cause and effect relationship between the protein kinase mutations and mdr expression. No changes in nuclear run-off assays could be detected, suggesting a non-transcriptional mechanism of regulation. Wild-type Y1 cells are more drug sensitive despite having higher levels of P-glycoprotein than the mutant cells. This paradoxical result may be explained by the higher rate of synthesis of steroids by the wild-type Y1 cells, which appear to be inhibitors of P-glycoprotein transport activity.

Cyclic AMP-dependent protein kinase controls basal gene activity and steroidogenesis in Y1 adrenal tumor cells.

Transfection of mouse Y1 adrenal tumor cells with DNA encoding mutant type I regulatory subunit generated stable transformants in which the basal activity of cAMP-dependent protein kinase was repressed. As expected, steroidogenesis in these kinase-deficient cells was no longer stimulated by corticotropin or cAMP analogues, and the expression of three cAMP-regulated genes (ornithine decarboxylase, urokinase-type plasminogen activator, and P450 side-chain cleavage) could no longer be induced. However, in addition to the loss of hormone responsiveness, the basal level of steroidogenesis and the constitutive expression of these cAMP-inducible genes was also repressed in kinase-defective mutant clones. To verify that functional cA-PK would revert this repressed phenotype, we transfected a cA-PK defective subclone of Y1 cells, Kin 8, with DNA encoding the C alpha and C beta subunits of cAMP-dependent protein kinase. Basal levels of steroid production were restored to normal in stable transformants, and the elevation of kinase activity following induction of the C-subunit expression vectors elicited a steroidogenic response. Gene transcription was also shown to be regulated by either C alpha or C beta as measured by the induction of plasminogen activator and ornithine decarboxylase mRNA levels and transcription rates. The dominant role played by cAMP-dependent protein kinase in these adrenal cells was demonstrated by experiments showing the regulation of ornithine decarboxylase gene expression by protein kinase C requires basal cAMP-dependent protein kinase activity.

Seasonality and toxigenicity ofVibrio cholerae non-01 isolated from different components of pond ecosystems of Dhaka City, Bangladesh

Diarrhoea due toVibrio cholerae non-01 is common in Bangladesh. Four hundred and eighty samples, including plants, water, phytoplankton and sediment, were collected from five ponds in Dhaka every 15 days for one year.V. cholerae non-01 was isolated from 181 (38%) of the samples. Two peaks were evident: one in April and the other in August/September. Forty-three (23%) of the 181 isolates were examined for toxigenicity and 19% were cytotoxic to Y1 adrenal cells. This study provides evidence of the likely infectious nature of some ponds and may have relevance to the epidemiology of diarrhoea caused byV. cholerae non-01 in Bangladesh.

The orphan receptor cDNA RDC4 encodes a 5-HT 1D serotonin receptor

The cDNA of RDC4, a putative receptor of the G protein-coupled receptor family, has been cloned by PCR methodology. The primary structure of this receptor showed homology with the serotonin 5-HT1A receptor. In this work, RDC4 mRNA has been injected in Y1 adrenal cells and Xenopus oocytes and RDC4 cDNA has been transfected transiently in cos-7 cells. In all these systems serotonin elicited a rise in cyclic AMP levels. Binding studies on membranes of the transfected cos-7 cells using [3H]-LSD showed a pattern of drug affinities consistent with the known properties of a 5-HT1D receptor. RDC4 therefore codes for a 5-HT1D receptor which in the studied systems is positively coupled to adenylate cyclase.

Embryonic chick heart expresses multiple muscarinic acetylcholine receptor subtypes. Isolation and characterization of a gene encoding a novel m2 muscarinic acetylcholine receptor with high affinity for pirenzepine.

Muscarinic acetylcholine receptors (mAChR) are G protein-coupled receptors which are highly conserved across mammalian species. Chick cardiac mAChR, however, have been shown to be pharmacologically, immunologically, and biochemically distinct from m2 mAChR expressed in mammalian heart. We previously reported the isolation and characterization of a novel chicken mAChR, cm4, which is expressed in chick heart and brain. We report here the isolation of an additional chicken mAChR gene whose deduced amino acid sequence is most homologous to the mammalian m2 receptor. Northern blot analysis demonstrated that this chicken m2 gene is also expressed in chick heart and brain. When stably transfected into Chinese hamster ovary (CHO) cells and Y1 adrenal carcinoma cells, the chicken m2 gene expresses a receptor protein which exhibits high affinity binding for the muscarinic antagonist quinuclidinyl benzilate and atropine, as well as the M1-selective antagonist pirenzepine and the M2-selective antagonist AF-DX 116. Therefore, when expressed in two heterologous cell lines, the chick m2 receptor has pharmacological properties that are similar to the chick m4 receptor as well as those reported for endogenous mAChR in chick cardiac cells. Consistent with the properties of the chick m4, as well as mammalian m2 and m4 receptors, the chick m2 receptor was able to functionally couple to both the inhibition of adenylate cyclase and the stimulation of phosphoinositide metabolism when expressed in CHO cells, but only the inhibition of adenylate cyclase when expressed in Y1 cells. We conclude from this study that the embryonic chick heart expresses multiple subtypes of mAChR which are highly conserved with their mammalian counterparts. Furthermore, the high degree of conservation between the mammalian m2 and the chick m2 muscarinic receptors suggests that the pharmacological differences that exist between these receptors are due to a relatively small number of specific amino acid changes rather than larger changes in receptor sequence or structure.

Evidence that an adrenal-specific nuclear protein regulates the cAMP responsiveness of the human CYP21B (P450C21) gene

Based on competition analysis of gel shift assays utilizing nuclear extracts from the mouse adrenal Y1 cell line, the cAMP-responsive element of the human CYP21B gene (-129/-96 base pairs (bp] is found to contain two overlapping nuclear protein binding elements. One, -126/-113 bp, interacts specifically with an adrenal-specific protein factor (ASP) while the other, -119/-110 bp, contains a GC boxlike sequence and binds Sp1. The nucleotide replacements (GG----CC at -113 and -112 bp) introduced within the sequence -129/-96 bp greatly decreased the binding affinities of both Sp1 and ASP to the fragment and resulted in a loss of cAMP-enhanced transcription of a reporter gene in transient transfection experiments. When the 14-bp ASP-binding sequence was inserted in front of the reporter gene, induced transcription by cAMP was observed that could be increased by multimerization of this -126/-113-bp sequence. The -126/-113-bp fragment revealed specific binding only with nuclear extracts from adrenal Y1 cells among various types of cells tested, indicating that the protein factor ASP is specifically expressed in adrenal cells. The nucleotide replacement (G----C at -117 bp) of the -126 -113-bp sequence not only abolishes the binding to ASP but also the cAMP-dependent transcription, strongly supporting the hypothesis that ASP is a tissue-specific transacting protein that directly functions as a transcription factor essential to the cAMP-dependent transcription. Furthermore, the -129/-115-bp sequence of the bovine cytochrome P450C21 gene corresponding to the human -126/-113-bp sequence is demonstrated to be functionally conserved with respect to both cAMP-dependent enhancement of transcription and ASP binding.

Regulation of CYP11A (P450SCC) and CYP17 (P450(17) alpha) gene expression in bovine luteal cells in primary culture

Cholesterol side chain cleavage cytochrome P450 (P450SCC) and 17 alpha-hydroxylase cytochrome P450 (P450(17) alpha) are key enzymes involved in steroid hormone biosynthesis. The 5'-flanking regions of the corresponding bovine genes have recently been characterized in the adrenal-derived Y1 cell line and specific DNA sequences, required for basal and cAMP-dependent gene expression, were identified. In order to investigate the molecular mechanisms controlling P450SCC and P450(17) alpha gene expression throughout the ovarian cycle, we devised an electroporation protocol to transfect bovine luteal cells in primary culture with specific chimeric DNA constructs. Transfection of such cells with reporter gene constructs containing 186 base pairs (bp) or more of the CYP11A (P450SCC) 5'-regulatory region resulted in cAMP-responsive reporter gene expression. Reporter gene constructs containing 101 bp or less of this regulatory region were expressed neither in the presence nor in the absence of forskolin. Thus, expression of the CYP11A gene in bovine luteal cells in primary culture appears to be controlled by a cis-acting element located between -186 to -101 bp. Transfection of bovine luteal cells in primary culture with reporter gene constructs containing increasing deletions of the bovine CYP17 (P450(17) alpha) 5'-regulatory region clearly demonstrated that none of the sequences were capable of promoting significant reporter gene expression, neither in the presence nor absence of forskolin. Since no 17 alpha-hydroxylase activity and no specific mRNA encoding P450(17) alpha have been detected in the bovine corpus luteum, the failure of expression of the P450(17) alpha reporter gene constructs mimics that of the endogenous gene. These results demonstrate that the regulation of CYP11A gene expression in bovine luteal cells is carried out by the same cis-acting element (-183/-101) which serves this role in the adrenal Y1 cell line. On the other hand, expression of the CYP17 gene in bovine luteal cells appears to be completely repressed, due to the absence of specific positive transcription factor(s) or the presence of negative regulatory factor(s). Thus, transfection experiments using bovine luteal cells in primary culture represent a first step towards the elucidation of molecular mechanisms underlying the regulation of cytochrome P450 gene expression throughout the ovarian cycle.

Expression of the human apolipoprotein E gene suppresses steroidogenesis in mouse Y1 adrenal cells.

The lipid transport protein, apolipoprotein E (apoE), is expressed in many peripheral tissues in vivo including the adrenal gland and testes. To investigate the role of apoE in adrenal cholesterol homeostasis, we have expressed a human apoE genomic clone in the Y1 mouse adrenocortical cell line. Y1 cells do not express endogenous apoE mRNA or protein. Expression of apoE in Y1 cells resulted in a dramatic decrease in basal steroidogenesis; secretion of fluorogenic steroid was reduced 7- to greater than 100-fold relative to Y1 parent cells. Addition of 5-cholesten-3 beta,25-diol failed to overcome the suppression of steroidogenesis in these cells. Cholesterol esterification under basal conditions, as measured by the production of cholesteryl [14C]oleate, was similar in the Y1 parent and the apoE-transfected cell lines. Upon incubation with adrenocorticotropin or dibutyryl cAMP, production of cholesteryl [14C]oleate decreased 5-fold in the Y1 parent cells but was unchanged in the apoE-transfected cell lines. These results suggest that apoE may be an important modulator of cholesterol utilization and steroidogenesis in adrenal cells.

Expression of the human apolipoprotein E gene suppresses steroidogenesis in mouse Y1 adrenal cells.

Expression of the human apolipoprotein E gene suppresses steroidogenesis in mouse Y1 adrenal cells.

RDC8 codes for an adenosine A2 receptor with physiological constitutive activity

The cDNA of an unidentified recently cloned G protein-coupled receptor, RDC8, has been expressed in Y1 adrenal cells, in dog thyrocytes in primary culture and in Xenopus oocytes. In all these systems this resulted in the activation of adenylyl cyclase and of the cyclic AMP cascade in the absence of any added external signal. However, this physiologically constitutive activator was inhibited by adenosine deaminase and by inhibitors of the adenosine A2 receptor. Cos 7 cells transfected with RDC8 cDNA constructs acquired binding characteristics of an adenosine A2 receptor. Moreover, RDC8 mRNA and adenosine A2 receptors display a very similar distribution in the brain. RDC8 therefore codes for an A2 adenosine receptor. Whether the physiologically constitutive activation of this receptor is entirely explained by endogeneously produced adenosine is as yet unknown.

Rat cholesterol side-chain cleavage cytochrome P-450 (P-450scc) gene. Structure and regulation by cAMP in vitro.

The entire rat P-450scc gene has been cloned, positions/sequences of the exon-intron boundaries (I-IX) are described and 940 base pairs (bp) of 5'-flanking DNA have been sequenced, compared to mouse, bovine and human genes, and analyzed by functional assays. Primer extension analysis mapped the transcription start site 32 bp upstream of the initiator methionine codon. The rat P-450scc promoter was ligated to the human growth hormone (GH) gene yielding p-940RsccGH. This rat P-450scc fusion gene and a mouse gene (p-1500MsccGH) were transiently transfected into primary cultures of rat granulosa cells and Y1 adrenal cells. In the Y1 cells primer extension analysis showed that the rat P-450sccGH gene was expressed at lower basal levels than that of the mouse gene but showed greater stimulation (4-8-fold) in response to 8-bromo-cyclic AMP than the mouse (3-4-fold). Similar results were obtained when the fusion genes were transfected into primary cultures of rat granulosa cells and production of GH was measured in the media of the cells stimulated with forskolin. Furthermore, we document that gonadotropins (follicle-stimulating hormone/luteinizing hormone) can induce luteinization of granulosa cells in vitro, that this process is associated with constitutive maintenance of P-450scc mRNA in the absence of hormones/cAMP, that these events associated with luteinization are differentiation-stage specific and occur only in granulosa cells of preovulatory follicles but not in small antral follicles, that the process can be inhibited by cycloheximide if the protein synthesis inhibitor is present during the first 6 h of exposure to luteinizing hormone but not if added for short durations (3-5 h) later, and that once luteinization is induced P-450scc mRNA expression and progesterone biosynthesis are not strictly dependent on cAMP. Thus, the P-450scc gene is regulated by both cAMP-dependent and cAMP-independent mechanisms, each of which are associated with a specific stage of granulosa cell differentiation. The DNA domains involved in regulating these two diverse processes remain to be determined. Although there was remarkable sequence homology among rat, mouse, bovine, and human genes within 70 bp of the transcriptional start site, no other sequence similarities revealed conserved functional domains among the four genes. Although some cAMP-responsive element-like sequences are present in the rat gene, these were not conserved in the other species; including the mouse which showed high sequence homology with the rat throughout 900 bp of 5'-flanking DNA. Thus, the cAMP domains specific to this and other steroidogenic genes remain to be clearly identified.

Tissue-Specific, Cyclic Adenosine 3′,5′-Monophosphate-lnduced, and Phorbol Ester-Repressed Transcription from the Human P450c17 Promoter in Mouse Cells

Cytochrome P450c17 is the single microsomal enzyme catalyzing steroid 17 alpha-hydroxylase and 17-20-lyase activities. It is expressed and regulated by tropic hormones in the human adrenal and gonads, but is not expressed in the placenta. To study the transcriptional regulation of the human P450c17 gene, we constructed 11 plasmids containing serial deletions of its 5' nontranslated region driving expression of the chloramphenicol acetyltransferase (CAT) reporter gene. These constructs were transfected into mouse adrenal Y1 and testis MA-10 cells and incubated with forskolin, 8-bromo-cAMP, or 12-O-tetradecanoyl-phorbol-13 acetate (TPA) for 12 h. Interpretation of results from standard constructions was difficult, apparently because some transcription was incorrectly initiated by DNA sequences in the vector. Therefore, we built a modified CAT reporter vector that eliminated detectable read-through transcription. In Y1 cells, the basal activity of constructs containing from -82 to -184 basepairs (bp) of 5' flanking DNA was between 80-150% of the promoterless control. Constructs containing at least -235 bp of this DNA expressed CAT at 540% of the control value, but addition of sequences to -774 had no further effect. Forskolin increased the expression of CAT activity to 300% above basal with constructions containing DNA from -184 to -774 bp. Constructs containing between -184 and -310 bp expressed CAT at 50% of the forskolin-induced levels in cells treated with TPA. Both basal and cAMP-induced expression were much lower in MA-10 cells than in Y1 cells and increased with increasing promoter length to -774.(ABSTRACT TRUNCATED AT 250 WORDS)

Human P450scc gene transcription is induced by cyclic AMP and repressed by 12-O-tetradecanoylphorbol-13-acetate and A23187 through independent cis elements.

Long-term regulation of mammalian steroid hormone synthesis occurs principally by transcriptional regulation of the gene for the rate-limiting cholesterol side-chain cleavage enzyme P450scc. Adrenal steroidogenesis is regulated primarily by two hormones: adrenocorticotropin, which works via cyclic AMP (cAMP) and protein kinase A, and angiotensin II, which works via Ca2+ and protein kinase C. Forskolin and 8-bromo-cAMP stimulated, while prolonged treatment with a phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA]) and a calcium ionophore (A23187) additively suppressed accumulation of endogenous P450scc mRNA in transformed murine adrenal Y1 cells. In Y1 cells transfected with 2,327 base pairs of the human P450scc promoter fused to the bacterial gene for chloramphenicol acetyltransferase (CAT), forskolin increased CAT activity 900% while combined TPA plus A23187 reduced CAT activity to 15% of the control level. Forskolin induced the P450scc promoter as rapidly as a promoter containing two cAMP-responsive elements fused to a simian virus 40 promoter, a system known to respond directly to cAMP. Basal expression was increased by sequences between -89 and -152 and was increased further by sequences between -605 and -2327. This upstream region also conferred inducibility by cAMP. TPA plus A23187 transiently increased CAT activity before repressing it, reflecting the complex actions of angiotensin II in vivo. Repression by prolonged treatment with TPA plus A23187 was mediated by multiple elements between -89 and -343. Induction of CAT activity by forskolin was not diminished by treatment with TPA plus A23187, nor were the regions of the promoter responsible for regulation by the two pathways coisolated. Thus, the human gene for P450scc is repressed by TPA plus A23187 by mechanisms and sequences independent of those that mediate induction by cAMP.