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

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

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

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

P450c11B3 mRNA, transcribed from a third P450c11 gene, is expressed in a tissue-specific, developmentally, and hormonally regulated fashion in the rodent adrenal and encodes a protein with both 11-hydroxylase and 18-hydroxylase activities.

The rat genome contains four P450c11 genes. One of these (CYP11B1) encodes P450c11 beta, which is the steroid 11 beta-hydroxylase found solely in the adrenal zona fasciculata/reticularis, and is responsible for the conversion of 11-deoxycorticosterone to corticosterone. A second P450c11 gene (CYP11B2) encodes P450c11AS, which is the aldosterone synthase found solely in the adrenal zona glomerulosa. P450c11AS has three activities, 11 beta-hydroxylase, 18-hydroxylase, and 18-oxidase, and is responsible for the conversion of 11-deoxycorticosterone to aldosterone. Recently, two more rat P450c11 genes, P450c11B3 and P450c11B4, were cloned. P450c11B4 appears to be a pseudogene, as two exons are replaced by unrelated DNA. P450c11B3 closely resembles P450c11 beta in mRNA and encoded amino acid sequences, predicting a protein of 498 amino acids. However, the expression of this mRNA and protein have not been demonstrated to date. We now demonstrate that this P450c11B3 mRNA is expressed in the adrenal gland several days after birth and is not expressed during fetal development or in the adult rat adrenal. Like P450c11 beta mRNA, P450c11B3 mRNA is expressed in the zona fasciculata/reticularis and not in the zona glomerulosa. However, the regulation of P450c11B3 mRNA expression is different from that of P450c11 beta mRNA, in that its abundance is decreased by ACTH in a sex-dependent fashion. Transfection of eukaryotic cells with a vector expressing P450c11B3 shows that this form of P450c11 can convert 11-deoxycorticosterone (DOC) to corticosterone and thus has the same enzymatic activity as P450c11 beta. In addition, P450c11B3 can convert DOC to 18-OH DOC and corticosterone to 18-OH corticosterone and thus has 18-hydroxylase activity similar to P450c11AS, but it lacks detectable 18-oxidase activity. Thus, P450c11B3 catalyzes 11 beta- and 18-hydroxylation and thus has a spectrum of activities midway between P450c11 beta and P450c11AS.

Transcriptional regulation of rat cytochrome P450c17 expression in mouse Leydig MA-10 and adrenal Y-1 cells: identification of a single protein that mediates both basal and cAMP-induced activities.

Cytochrome P450c17, 17 alpha-hydroxylase/17,20 lyase, is a key enzyme in the steroidogenic pathway leading to the production of corticosteroids and androgens from the adrenal gland and sex steroids from the gonads. Both enzymatic activities of the protein are encoded by a single gene, CYP17, which is expressed in both the human adrenal and gonad but not in the placenta, and in the rodent gonad and placenta but not the rodent adrenal. We isolated and sequenced a full-length rat genomic clone (7,553 bases) containing the entire coding region of the rat P450c17 gene, and all intronic sequences and 1,560 bp of 5'-flanking DNA (EMBL Acc#X69816). To determine which sequences in the rat P450c17 promoter may be responsible for basal and cAMP-stimulated gene transcription, deletion constructs containing between -1,560 and -53 base pairs of 5'-flanking DNA from the rat P450c17 gene were ligated to plasmids expressing the reporter gene luciferase and transfected into two mouse cell lines, adrenal Y-1 cells, and testicular Leydig MA-10 cells. Highest basal and cAMP-stimulated luciferase activity were found in constructions containing 156 bp of 5'-flanking DNA. This construction contains a sequence very similar to the consensus cis element reported to be responsible for cAMP enhancement of the rat somatostatin gene and also overlaps a sequence similar to the consensus element for the orphan steroid receptor SF-1. Gel mobility-shift analysis, using a 30-bp oligonucleotide containing this region incubated with cellular extracts from cultured mouse adrenal Y-1 and mouse Leydig MA-10 cells, revealed all the extracts to contain two proteins that bind to this sequence. Neither DNA-protein complex was further retarded by co-incubation with an anti-CREB antibody, suggesting that cAMP regulation of this gene occurs via a non-CREB protein. Mutation of this oligonucleotide resulted in loss of binding of only one of these proteins, but resulted in loss of both basal and cAMP stimulation of rat P450c17 promoter-regulated gene transcription. Southwestern analysis suggests that one of these proteins is larger than SF-1. This study suggests that a protein that binds to an SF-1 like sequence regulates both basal and cAMP-stimulated rat P450c17 gene expression in rodent cells.

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

Studies of adrenal steroidogenesis have been facilitated by the availability of immortalized mouse adrenocortical Y-1 cells. We sought to make new, alternative mouse steroidogenic cell lines by genetically targeted tumorigenesis. Transgenic mice were constructed expressing both the SV40 T-antigen and a bacterial neomycin-resistance gene under the control of the promoter for the human P450 cholesterol side-chain cleavage (P450scc) gene, which encodes the first and rate-limiting enzyme in steroidogenesis. Two female transgenic mice expressed T-antigen in various nonsteroidogenic tissues but generated tumors only in the adrenals, suggesting adrenal tumor formation was an early event. Ovarian tissues, which, unlike the adrenal, do not make steroids in fetal or early postnatal life, did not develop tumors. Cell lines derived from the adrenal tumors were resistant to the neomycin analog G418. Clonal sublines are stable, growing easily in monolayers with a doubling time of 24-60 h. The cell lines secrete progesterone and 11-deoxycorticosterone, indicating these cells express the P450scc system, 3 beta-hydroxysteroid dehydrogenase, and 21-hydroxylase activity. However the 21-hydroxylase activity was not mediated by P450c21, as the cells lacked P450c21 mRNA. The cells did not secrete any 11-hydroxylated steroids, although they contained P450c11 beta mRNA. Both the secretion of progesterone and the abundance of P450scc mRNA increase in response to 8-bromo-cAMP, but not to ACTH or angiotensin II. In addition to expression of steroidogenic enzyme mRNAs, one cell line also expresses mouse renin-1 mRNA, making these cells useful for studies of the role of adrenal renin in regulating adrenal steroidogenesis. These findings represent an approach in transgenic mice to develop highly differentiated adrenal cell lines.