Evan R. Simpson

Brain aromatase cytochrome P-450 messenger RNA levels and enzyme activity during prenatal and perinatal development in the rat

Aromatase cytochrome P-450 (P-450AROM) enzyme activity catalyzes the conversion of androgens to estrogens in specific brain areas. During development local estrogen formation is thought to influence the sexual differentiation of neural structures (i.e. increase neurite growth and establish neural circuitry) and modulate reproductive functions. This study was undertaken to investigate the ontogeny of the (P-450AROM) enzyme and its messenger RNA (mRNA) in medial basal hypothalamic (MBH) and preoptic area (POA) tissue during late fetal and perinatal development of the rat. Aromatase activity in the MBH-POA was negligible before gestational day (GD) 16 (< 0.1 pmol/h/mg protein), increased over 10-fold at GD 17 and continued to increase (over 5-fold) to peak levels at GD 19 (> 5.0 pmol/h/mg protein), and then declined to low levels at GD 22 and 2 days post-birth (approximately 1 pmol/h/mg protein). The profile of P-450AROM mRNA in the MBH-POA tissue was characterized by a predominant 2.7 kilobase (kb) mRNA species, similar in size to the largest functional P-450AROM mRNA observed in adult rat ovarian tissue. At GD 15, the P-450AROM mRNA was undetectable; low but detectable levels were seen at GD 17, the abundance increased at later time points and remained at peak levels on GDs 18 through 20, decreased slightly by GD 22, and then declined further by 2 days post-birth. The developmental increase in P-450AROM mRNA levels correlated with the ascending pattern of enzyme activity before GD 19, but the marked decrease in enzyme activity seen after GD 19 was not accompanied by a corresponding decline in mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the biosynthesis of cytochromes P-450 involved in steroid hormone synthesis.

The actions of ACTH to regulate the synthesis of the various enzymes involved in steroid hormone biosynthesis have been studied using bovine adrenocortical cells in monolayer culture. ACTH causes an increase in the synthesis of both the mitochondrial and the microsomal forms of cytochrome P-450 involved in steroid hormone biosynthesis, as well as of the iron-sulfur protein involved in transferring electrons to the mitochondrial forms of cytochrome P-450, namely, adrenodoxin. This increased synthesis is reflective of an increase in translatability of mRNA species specific for these various proteins, and appears in each case to be mediated by cyclic AMP. Whereas the mitochondrial proteins are synthesized as precursors of higher molecular weight which are processed upon insertion into the mitochondria, the microsomal proteins are synthesized as species identical in molecular weight to the mature forms. In order to determine whether the action of ACTH to increase the rate of synthesis of these proteins is the result of an increase in the levels of specific mRNA species, cDNA clones complementary to these mRNA species are being isolated. These probes will also make it possible to characterize the genes encoding the steroidogenic enzymes, as well as to identify regulatory elements which control their transcription.

Cholesterol metabolism in cancer cells in monolayer culture. VI. Metabolism of high-density lipoprotein

Abstract The metabolism of high-density lipoprotein (HDL) in cells of five human cancer cell lines maintained in monolayer culture was investigated. In cells of some of the lines there was evidence of high-affinity binding sites for HDL, whereas in others this could not be demonstrated. However, in one cell line, viz., HEC-B-296 (human endometrial carcinoma), degradation of the protein component of HDL was demonstrated. The proteolytic activity was specific for HDL in so far as human serum albumin was not degraded by these cells. However, this degradative process did not involve internalization of the HDL molecule and degradation was not mediated by lysosomal proteolytic enzymes. HDL, when present in the medium, did not affect the degradation of low-density lipoprotein and low-density lipoprotein did not affect the degradation of HDL. HDL did not affect significantly cholesterol biosynthesis or cholesteryl ester biosynthesis as estimated from the activity of the regulatory enzymes, 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl-CoA:cholesterol acyltransferase. The degradation of HDL by HEC-B-296 cells was inhibited, to various degrees, when trypsin inhibitor or a protease inhibitor such as leupeptin, was present in the culture medium. It is concluded that degradation of the protein component of HDL by human neoplastic cells of the HEC-B-296 line was the result of activity of a proteolytic enzyme that is present on the external surface of the cells.

Cell-free synthesis of precursor forms of mitochondrial steroid hydroxylase enzymes of the bovine adrenal cortex☆

Abstract Newly synthesized, [ 35 S]methionine-labeled cholesterol side-chain cleavage cytochrome P -450, 11β-hydroxylase cytochrome P -450, adrenodoxin, and adrenodoxin reductase were immunoisolated from radiolabeled bovine adrenocortical cells and from rabbit reticulocyte lysate translation systems programmed with bovine adrenocortical RNA. Cholesterol side-chain cleavage cytochrome P -450 immunoisolated from a reticulocyte lysate translation system had an apparent molecular weight of 54,500 whereas this cytochrome P -450 immunoisolated from radiolabeled bovine adrenocortical cells had an apparent molecular weight of 49,000, an apparent molecular weight identical to that of the purified protein. Similarly, newly synthesized, [ 35 S]methionine-labeled 11β-hydroxylase cytochrome P -450 immunoisolated from a reticulocyte lysate translation system had an apparent molecular weight 5500 daltons larger than that immunoisolated from radiolabeled adrenocortical cells (48,000) and the authentic cytochrome (48,000). The cell-free translation products of adrenodoxin and adrenodoxin reductase were also several thousand daltons larger than the corresponding mitochondrial proteins. The apparent molecular weight of adrenodoxin immunoisolated from a reticulocyte lysate translation system was 19,000, while that of the authentic protein was 12,000. Adrenodoxin reductase immunoisolated from a lysate translation system had an apparent molecular weight of 53,400; an apparent molecular weight 2300 daltons larger than that of adrenodoxin reductase immunoisolated from radiolabeled adrenocortical cells or purified by conventional techniques. These results demonstrate that all of the components of the mitochondrial steroid hydroxylase systems of the bovine adrenal cortex are synthesized as precursor molecules of higher molecular weight. Presumably, the precursor proteins are post-translationally converted to the mature enzymes upon insertion into the mitochondrion by a process which includes the proteolytic cleavage of the precursor segments.

Sequencing of cDNA inserts encoding aromatase cytochrome P-450 (P-450AROM)

Two cDNA inserts complementary to mRNA encoding aromatase cytochrome P-450 (P-450AROM) have been isolated and characterized by restriction mapping and sequencing. The overlapping sequence encoded by these inserts is identical, and a putative heme-binding region has been identified. In addition, the open reading frame contains the sequences of all four cysteine-containing tryptic peptides isolated by Chen et al. (1986) from purified cytochrome P-450AROM. The inserts differ in the use of alternative poly A-addition signals, which is consistent with the presence of two major species of mRNA in human placenta, of 3.0 and 2.4 kb, which hybridize to these inserts. The identity of sequence between the two inserts and the likely presence of alternative poly A-addition signals, is suggestive that only one form of cytochrome P-450AROM is encoded by these mRNA species.

Regulation of Synthesis and Activity of Cytochrome P-450 Enzymes in Physiological Pathways

Regulation of the levels and activities of various forms of cytochrome P450 has important implications in human biology. For example, it is well known that different individuals have different capacities to metabolize various drugs.1 Such variation may result, in part, from an allelic distribution of P-450 isozymes;2 or it may also result from individual variations in the level of a specific isozyme.3,4 Also, deficiencies of different steroid hydroxylases lead to various disease states. For example, adrenal hyperplasia occurs in approximately one in 5000 births, making it one of the more common inborn errors of metabolism.5 In at least 95% of these cases, the deficiency is detected as a decrease in steroid 21-hydroxylation in the adrenal cortex,6 a reaction catalyzed by a specific form of P-450 (P-450C21), leading to deficient cortisol biosynthesis. These are but a few examples which illustrate the reasons why investigation of the regulation of P-450 activities has become such an active area of research. In each example, understanding of the molecular basis of variations in P-450 gene expression will be necessary for the complete elucidation of the different phenotypes.

Regulation of cytochrome P-450c by glucocorticoids and polycyclic aromatic hydrocarbons in cultured fetal rat hepatocytes

The actions of polycyclic aromatic hydrocarbons and glucocorticoids to regulate the synthesis of cytochrome P-450c (the major isozyme induced by polycyclic aromatic hydrocarbons) were investigated in fetal rat hepatocytes maintained in primary monolayer culture. Treatment of hepatocytes in culture with 1,2-benzanthracene resulted in a 50-fold increase in 7-ethoxycoumarin O-deethylase activity. The level of P-450c increased in the cells in a time-dependent fashion as determined by immunoelectrophoretic analysis. The inductive effect of BA was potentiated approximately 1.6- to 2.3-fold when 1 microM dexamethasone was included in the culture medium. However, dexamethasone alone had little or no effect on the induction of P-450c. The rate of synthesis of P-450c was examined by immunoisolation of the specific isozyme from total cellular proteins radiolabeled with [35S]methionine and from the protein products formed during in vitro translation of the isolated mRNA. In addition, the amount of mRNA specific for cytochrome P-450c was determined by Northern blot analysis of RNA extracted from cultured cells. The changes in the rates of synthesis and mRNA levels were found to parallel the changes in enzyme activity. The concentration of dexamethasone required to cause a half-maximal increase in P-450c content in the presence of 1,2-benzanthracene was between 10(-8) and 10(-7) M. It is concluded that glucocorticoids act synergistically with polycyclic aromatic hydrocarbons to increase the levels of P-450c expressed in the fetal rat liver, and that this action is likely mediated by the classical type II glucocorticoid receptor.

Induction of 17α-hydroxylase (cytochrome P-45017α) activity by adrenocorticotropin in bovine adrenocortical cells maintained in monolayer culture☆

Abstract Using bovine adrenocortical cells in monolayer culture it has been shown that treatment with adrenocorticotropin (ACTH) causes a dramatic increase in 17α-hydroxylase activity. In postmitochondrial supernatant fractions (PMS) prepared from cells maintained in culture, there was a 15-fold increase in 17α-hydroxylase activity 36 h following initiation of ACTH treatment compared with the activity measured in PMS prepared from control cells. In the continued presence of ACTH, 17α-hydroxylase activity declined; however, even after 60 h of exposure to ACTH, 17α-hydroxylase activity was eight times higher than that present in control cells. The dramatic increase in 17α-hydroxylase activity provides an explanation for the previously observed phenomenon that following initiation of ACTH treatment of bovine adrenocortical cells in monolayer culture there is a shift in the pattern of corticosteroid secretion from approximately equal amounts of cortisol and corticosterone to almost exclusively cortisol. Thus, the modulation of 17α-hydroxylase activity by ACTH action appears to serve a key regulatory role in the pattern of corticosteroid production. Soluble cytosolic factors apparently do not participate in the regulation of 17α-hydroxylase activity in the bovine adrenal cortex. Increases in the magnitude of substrate-induced absorbance changes are indicative that the increase in 17α-hydroxylase activity is due, at least in part, to an elevation of cytochrome P -450 17 α synthesis.

Regulation of aromatase activity of cultured adipose stromal cells by catecholamines and adrenocorticotropin

Adipose tissue is the major site of estrogen formation in postmenopausal women. We have previously reported (Simpson, E.R., Ackerman, G.E., Smith, M.E. and Mendelson, C.R. (1981) Proc. Natl. Acad. Sci. (U.S.A.) 78, 5690-5694; Mendelson, C.R., Cleland, W.H., Smith, M.E. and Simpson, E.R. (1982) Endocrinology 111, 1077-1085) that aromatase activity of human adipose stromal cells in culture is stimulated by glucocorticoids and by dibutyryl cyclic AMP (Bt2-cAMP). In order to establish which physiological factors might stimulate aromatase activity of these cells by activation of adenylate cyclase, we have investigated the roles of adrenocorticotropin (ACTH) and isoproterenol to increase cyclic AMP levels and stimulate the aromatization of androstenedione. In the presence of methylisobutylxanthine (MIX), ACTH stimulated cyclic AMP formation and aromatase activity in a time- and concentration-dependent manner. The concentration of ACTH required for half-maximal stimulation was approximately 10(-8) M. Isoproterenol, in the presence of MIX, stimulated cyclic AMP formation in a time- and concentration-dependent fashion, and also stimulated aromatase activity. These effects of isoproterenol appeared to be mediated by binding of the agonist to a population of beta-adrenergic receptors. On the basis of these and our previous studies, we suggest that ACTH may play an important role in stimulating estrogen formation by human adipose tissue, both directly, and by stimulating the adrenal cortex to produce both substrate, androstenedione, and inducing agent, namely cortisol.

Ontogeny of adrenal steroid hydroxylases: evidence for cAMP-independent gene expression

Total RNA from normal and anencephalic human fetal adrenals was examined by blot analysis for transcripts encoding P-450scc, P-450(11) beta, P-450(17) alpha, P-450C21 and adrenodoxin using bovine cDNA clones specific for these different enzymes. The specific contents of RNA encoding these components of the adrenocortical steroidogenic pathway were found to be similar in both types of adrenal tissue. Likewise, immunoblot analysis showed comparable concentrations of P-450scc, P450(17) alpha and adrenodoxin protein to be present in adrenal tissues from normal and anencephalic human fetuses. Immunoblot analysis of homogenates of fetal sheep adrenals of increasing gestational age (85-145 days) showed constant levels of P-450scc and P-450(11) beta, but increasing P-450(17) alpha content, especially near term. Both sheep fetuses prior to 136 days gestational age and human anencephalic fetuses are known to have extremely low circulating levels of immunoreactive ACTH as well as very low adrenal adenylate cyclase activity. Thus, it is concluded that factors other than pituitary ACTH which operate independent of adenylate cyclase activation are required for the initial expression (imprinting) of steroid hydroxylase genes.