The causal relationship between mutations in cAMP-dependent protein kinase and the loss of adrenocorticotropin-regulated adrenocortical functions.

The Y1 adrenocortical tumor cell mutants, Kin-7 and Kin-8, harbor point mutations in the regulatory subunit (RI) of the type 1 cAMP-dependent protein kinase (cAMPdPK) that render the enzyme resistant to activation by cAMP. These mutants also are resistant to many of the regulatory effects of ACTH and cAMP. In order to examine the causal relationships between the mutations in cAMPdPK and the resistance to ACTH and cAMP, the Kin mutants were transfected with expression vectors encoding wild type subunits of cAMPdPK in order to restore cAMP-responsive protein kinase activity. The transformants then were screened for the concomitant recovery of cellular responsiveness to ACTH and cAMP. In the mutant Kin-7, cAMP-responsive protein kinase activity was recovered after transfection with an expression vector encoding wild type mouse RI. Protein kinase activity in the mutant Kin-8 remained largely cAMP-resistant after transfection with the RI expression vector but could be rendered cAMP-responsive by transfection with an expression vector encoding the wild type catalytic subunit. The recovery of cAMP-responsive protein kinase activity was accompanied by the recovery of steroidogenic and morphological responses to ACTH and cAMP, suggesting that the cAMP-dependent signaling cascade plays an obligatory role in these actions of ACTH. The growth-regulatory effects of cAMP were not reversed with the recovery of cAMP-responsive protein kinase activity, suggesting that cAMP-resistant growth regulation results from second-site, adaptive mutations either in the original Kin mutant population or in the transformants. Studies on the conversion of 22(R)-hydroxycholesterol into steroid products in parent and mutant cells indicate that the Kin mutations reduce the steroidogenic capacity of the cell as well as inhibit the hormone- and cyclic nucleotide-dependent mobilization of substrate cholesterol.     

Recovery of cyclic nucleotide regulation in protein-kinase-defective adrenal cells through somatic cell fusion

A mutant cell line (designated Kin-8), isolated from the Y1 mouse adrenocortical tumor cell line on the basis of its resistance to growth-inhibition by 8-bromoadenosine 3', 5'-monophosphate (8BrcAMP), was resistant to the steroidogenic effects of the cyclic nucleotide analog and did not round up in the presence of 8BrcAMP as did responsive Y1 adrenal cells. In Kin-8 cells, the mutation to cyclic nucleotide resistance was associated with a defective type 1 cAMP-dependent protein kinase activity, suggesting an obligatory role for the enzyme in the regulation of these adrenal functions. In this study, the Kin-8 mutant was fused with a rat glioma cell line (C6) in order to analyze the genetic behavior of the protein kinase mutation in somatic cell hybrids. The growth of C6 glial cells was inhibited by 8BrcAMP and its cAMP-dependent protein kinase responded normally to cAMP. In addition, C6 cells had no capacity for steroidogenesis nor did they round up when treated with 8BrcAMP. In Kin-8 X C6 hybrids, the protein kinase mutation seemed to behave recessively. The growth of hybrid cells was inhibited by 8BrcAMP and the protein kinase responded to cAMP over a normal range. Kin-8 X C6 hybrids, when treated with 8BrcAMP, exhibited steroidogenic activities which were greater than the activity measured in either fusion partner and which had lower ED50 values for 8BrcAMP. In addition, Kin-8 X C6 hybrids rounded up in the presence of 8BrcAMP, a morphologic change unlike that seen with either fusion partner. The effects of 8BrcAMP on the steroidogenic activity and morphology of Kin-8 X C6 hybrids was reminiscent of the effects of the cyclic nucleotide on cAMP-responsive, parental Y1 adrenal cells. These results suggest that cell fusion provided a normal cAMP-dependent protein kinase for Kin-8 cells and led to the recovery of a cAMP-responsive adrenal phenotype. type. These results provide additional evidence in support of an obligatory role for cAMP-dependent protein kinase in the regulation of adrenal steroidogenesis, cell division, and cell shape.     

Cholesterol side-chain cleavage, cytochrome P450, and iron-sulfur protein in human placental mitochondria.

Cytochrome P450 and the associated iron-sulfur protein have been characterized in human placental mitochondria by means of optical absorbance difference spectrophotometry and electron paramagnetic resonance spectrometry. These two proteins occur in a molar ratio of about 1:1 in human placental mitochondria, and the cytochrome P450 appears to be that form involved in cholesterol side-chain cleavage. Pregnenolone formation from endogenous mitochondrial cholesterol, as measured by radioimmunoassay, follows a biphasic time-course similar to the situation in other steroidogenic tissues. The specific activity of cholesterol side-chain cleavage, and the specific contents of cytochrome P450 and the iron-sulfur protein in the mitochondria, are 2- to 3-fold higher at term than in the 1st and 2nd trimesters. When expressed in terms of the cytochrome P450 content, the rate of pregnenolone formation is high, suggesting that cholesterol side-chain cleavage in human placenta is in an activated state.     

Promoter elements of the mouse 21-hydroxylase (Cyp-21) gene involved in cell-selective and cAMP-dependent gene expression

Cyp-21 (the mouse steroid 21-hydroxylase gene) is expressed exclusively in cells of the adrenal cortex, is induced by ACTH and cAMP, and is required for corticosteroid synthesis. This review examines the molecular basis for the regulated expression of Cyp-21 in the ACTH-responsive, mouse adrenocortical tumor cell line, Y1. We demonstrate that 330 bp of 5′-flanking DNA from the Cyp-21 gene are sufficient for cell-selective and ACTH-induced expression of Cyp-21, and that this promoter region comprises multiple, closely spaced enhancer elements each of which is required for promoter function. Within this promoter, we define three related elements that contain variations of an AGGTCA motif and that contribute to the cell-selective expression of Cyp-21. Variations of these same AGGTCA-bearing elements are also involved in the expression of Cyp 11a and Cyp 11b in Y1 adrenocortical cells. These elements interact with the same or closely related nuclear proteins found only in steroidogenic cell lines. Taken together, these results suggest that shared elements contribute to the adrenal cell-selective expression of at least three steroidogenic cytochrome P450 genes.

The element at −170 and the related elements at −65, −140 and −210 in the Cyp-21 promoter are not active as enhancers in the mutant Y1 cell line, Kin-8. Kin-8 cells contain a mutation in the regulatory subunit of the type 1 cAMP-dependent protein kinase that renders the enzyme resistant to activation by cAMP. Therefore, these elements appear to be selectively dependent upon an intact cAMP-dependent protein kinase for enhancer function. Individually, none of these elements confer cAMP-dependence to a reporter gene driven by a heterologous promoter. On the basis of these observations, we suggest that ACTH- and cAMP-dependent expression of Cyp-21 requires the combined actions of the element at −170, and the related elements at −140, −210 and −65.     

Conditionally immortalized adrenocortical cell lines at undifferentiated states exhibit inducible expression of glucocorticoid-synthesizing genes.

To facilitate studies on differentiation of adrenocortical cells and regulation of steroidogenic genes, we established cell lines from adrenals of adult transgenic mice harboring a temperature-sensitive large T-antigen gene of simian virus 40. Adrenal glands of the mice exhibited normal cortical zonation including a functionally undifferentiated cell-layer between the aldosterone-synthesizing zona glomerulosa cells and the corticosterone-synthesizing zona fasciculata cells. At a permissive temperature (33 degrees C), established cell lines AcA201, AcE60 and AcA101 expressed steroidogenic genes encoding steroidogenic factor-1, cholesterol side-chain cleavage P450scc, and steroidogenic acute regulatory protein, which are expressed throughout adrenal cortices and gonads. Genes encoding 3 beta-hydroxysteroid dehydrogenase and steroid 21-hydroxylase P450c21, which catalyze the intermediate steps for syntheses of both aldosterone and corticosterone, were inducible in the three cell lines in temperature- and/or dibutyryl cAMP-dependent manners. Notably, these cell lines displayed distinct expression patterns of the steroid 11 beta-hydroxylase P45011 beta gene responsible for the zone-specific synthesis of corticosterone. AcA201 cells expressed the P45011 beta gene at 33 degrees C, showing the property of the zona fasciculata cells, while AcE60 cells expressed it upon a shift to a nonpermissive temperature (39 degrees C). On the other hand, AcA101 expressed the P45011 beta gene at 39 degrees C synergistically with exposure to dibutyryl cAMP. None of these clones express the zona glomerulosa-specific aldosterone synthase P450aldo gene under the conditions we tested. These results show that AcE60 and AcA101 cells display a pattern of the steroidogenic gene expression similar to that of the undifferentiated cell-layer and are capable of differentiating into the zona fasciculata-like cells in vitro.     

mRNA from mutant Y1 adrenal cells directs the synthesis of altered regulatory subunits of type 1 cAMP-dependent protein kinase

The molecular basis for altered cyclic AMP-dependent protein kinase activity was examined in three different mutant clones (Kin-1, Kin-7, and Kin-8) derived from the Y1 mouse adrenocortical cell line. Parental Y1 cells and the Kin mutants were labeled with L-[35S] methionine and the regulatory subunit of the type 1 cAMP-dependent protein kinase isozyme (RI) was immunoprecipitated from each clone with a specific guinea pig antiserum. When analyzed by electrophoresis on isoelectric focusing gels, the immunoprecipitates from mutant clones exhibited parental forms of RI plus an additional acidic variant form which likely accounted for altered cAMP-dependent protein kinase activity. Poly(A+) RNA was isolated from Y1 and Kin mutant cells and was translated in a cell-free, reticulocyte lysate system in the presence of L-[35S]methionine. The RI synthesized from poly(A+) RNA was immunoprecipitated from the translation mixture and analyzed on isoelectric focusing gels. The poly(A+) RNA from the Kin mutant clones directed the synthesis of parental and acidic variant forms of RI. These results suggest that the altered electrophoretic forms of RI arise from mutations in one of two RI genes rather than from post-translational modifications of the protein. The coexistence of parental and variant forms of RI in the Kin mutants indicate that the mutations are codominant.     

Purification of cytochrome P-450 from bovine adrenocortical mitochondria

One soluble cytochrome P.450 from bovine adrenocortical mitochondria has been purified to near homogeneity. The purified enzyme catalyses side-chain cleavage of cholesterol and to a much lesser extent 11β-hydroxylation (<13% side-chain cleavage) but shows no 18-hydroxylase activity. The molecular weight of this P.450 is approximately 800,000.     

The insulin-like growth factor, somatomedin C, induces the synthesis of cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin in ovarian cells

The actions of insulin and somatomedin C (insulin-like growth factor I) on cholesterol side-chain cleavage activity and the synthesis of cytochrome P-450scc and adrenodoxin were investigated in primary cultures of swine ovarian (granulosa) cells. Nanomolar concentrations of pure human somatomedin C stimulated biosynthesis of progesterone and 20 alpha-hydroxypregn-4-en-3-one. Moreover, in the presence of exogenous sterol substrate for cholesterol side-chain cleavage, somatomedin C significantly enhanced pregnenolone biosynthesis in a time- and dose-dependent manner. This augmentation of functional cholesterol side-chain cleavage activity was accompanied by a dose-dependent (2-16-fold) increase in [35S]methionine incorporation into specific immunoprecipitable cytochrome P-450scc and adrenodoxin. Micromolar concentrations of insulin (but not proinsulin or desoctapeptide) also induced synthesis of cholesterol side-chain cleavage constituents by 4-7-fold. These results demonstrate that an insulin-like growth factor, somatomedin C, exerts discrete differentiating effects on ovarian cells characterized by increased synthesis of immunospecific cytochrome P-450scc and adrenodoxin. Thus, we infer that somatomedin C may serve a critical role in the differentiation of steroidogenic cells in the mammalian ovary.     

Significantly increased cortisol secretion in normal adrenocortical cells transfected with K-ras mutants derived from human functional adrenocortical tumors

Our previous study showed that the mutation hotspots of the K-ras proto-oncogene in human functional adrenocortical tumors are in codons 15, 16, 18, and 31, thus differing from the sites in other tumors. In addition, analyzing the K-Ras protein by a recombinant DNA technique showed that the activity of endogenic GTPase and the GTPase-activating protein (GAP)-binding ability were significantly decreased in patients with these tumors. The aim of this study was to understand whether those K-ras mutants, which were found only in human adrenocortical tumors, play an important role in these tumors. Thus, the mutant K-ras cDNA was constructed with mammalian expression vectors and transfected into normal adrenocortical cells. The amount of cortisol secreted by the transfected cells was 20 to 30 times that of normal cells. Furthermore, Northern blot analysis revealed that the expression of the three steroidogenesis-related genes P450(scc) (cholesterol side-chain cleavage enzyme), P450(C17) (17alpha-hydroxylase/17, 20-lyase), and P450(C21) (steroid 21-hydroxylase) gene increased in the transfected cells. The K-ras oncogene significantly increases cortisol secretion by normal adrenocortical cells.     

A novel pathway for sequential transformation of 7-dehydrocholesterol and expression of the P450scc system in mammalian skin.

Following up on our previous findings that the skin possesses steroidogenic activity from progesterone, we now show widespread cutaneous expression of the full cytochrome P450 side-chain cleavage (P450scc) system required for the intracellular catalytic production of pregnenolone, i.e. the genes and proteins for P450scc enzyme, adrenodoxin, adrenodoxin reductase and MLN64. Functionality of the system was confirmed in mitochondria from skin cells. Moreover, purified mammalian P450scc enzyme and, most importantly, mitochondria isolated from placenta and adrenals produced robust transformation of 7-dehydrocholesterol (7-DHC; precursor to cholesterol and vitamin D3) to 7-dehydropregnenolone (7-DHP). Product identity was confirmed by comparison with the chemically synthesized standard and chromatographic, MS and NMR analyses. Reaction kinetics for the conversion of 7-DHC into 7-DHP were similar to those for cholesterol conversion into pregnenolone. Thus, 7-DHC can form 7-DHP through P450scc side-chain cleavage, which may serve as a substrate for further conversions into hydroxy derivatives through existing steroidogenic enzymes. In the skin, 5,7-steroidal dienes (7-DHP and its hydroxy derivatives), whether synthesized locally or delivered by the circulation, may undergo UVB-induced intramolecular rearrangements to vitamin D3-like derivatives. This novel pathway has the potential to generate a variety of molecules depending on local steroidogenic activity and access to UVB.     

Levels of messenger ribonucleic acid encoding cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor in bovine follicles and corpora lutea throughout the ovarian cycle

To investigate the molecular basis for the pattern of ovarian steroid production during the bovine estrous cycle, the relative levels of mRNA specific for cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor were determined in ovarian antral follicles of differing size (less than 3-18 mm) and corpora lutea from the early, early-mid, late-mid, and regressionary stages. Total and poly(A)+ RNA was size-fractionated on agarose-formaldehyde gels, transferred to nylon filters and hybridized to specific 32P-labeled probes. The levels of mRNAs for the rate-limiting enzymes in the conversion of cholesterol into progesterone, namely cholesterol side-chain cleavage cytochrome P-450 and its electron donor, adrenodoxin, were higher in corpora lutea than in follicles. Conversely the levels of mRNA specific for the key regulatory enzyme in the conversion of pregnenolone or progesterone to androgen, namely 17 alpha-hydroxylase cytochrome P-450, were high in all antral follicles examined but were low in young corpora lutea and undetectable in more mature corpora lutea. Low density lipoprotein receptor mRNA was detectable in antral follicles and corpora lutea but the levels were greater in corpora lutea. These results suggest that the pattern of changes in steroid hormone biosynthesis during the bovine estrous cycle and in the ovarian content of steroidogenic enzymes is related to and probably dependent upon the pattern of change in levels of mRNAs for steroidogenic enzymes and related proteins.     

Developmental and hormonal regulation of murine scavenger receptor, class B, type 1.

The scavenger receptor, class B, type I (SR-BI), is the predominant receptor that supplies plasma cholesterol to steroidogenic tissues in rodents. We showed previously that steroidogenic factor-1 (SF-1) binds a sequence in the human SR-BI promoter whose integrity is required for high-level SR-BI expression in cultured adrenocortical tumor cells. We now provide in vivo evidence that SF-1 regulates SR-BI. During mouse embryogenesis, SR-BI mRNA was initially expressed in the genital ridge of both sexes and persisted in the developing testes but not ovary. This sexually dimorphic expression profile of SR-BI expression in the gonads mirrors that of SF-1. No SR-BI mRNA was detected in the gonadal ridge of day 11.5 SF-1 knockout embryos. Both SR-BI and SF-1 mRNA were expressed in the cortical cells of the nascent adrenal glands. These studies directly support SF-1 participating in the regulation of SR-BI in vivo. We examined the effect of cAMP on SR-BI mRNA and protein in mouse adrenocortical (Y1-BS1) and testicular carcinoma Leydig (MA-10) cells. The time courses of induction were strikingly similar to those described for other cAMP- and SF-1-regulated genes. Addition of lipoproteins reduced SR-BI expression in Y1-BS1 cells, an effect that was reversed by administration of cAMP analogs. SR-BI mRNA and protein were expressed at high levels in the adrenal glands of knockout mice lacking the steroidogenic acute regulatory protein; these mice have extensive lipid deposits in the adrenocortical cells and high circulating levels of ACTH. Taken together, these studies suggest that trophic hormones can override the suppressive effect of cholesterol on SR-BI expression, thus ensuring that steroidogenesis is maintained during stress.