regulation of steroid hydroxylase gene expression: importance to physiology and disease

Pharmac. Ther. Vol. 58, pp. 301-317, 1993 0163-7258/93 $24.00 Printed in Great Britain. All rights reserved © 1993 Pergamon Press Ltd

Associate Editor: D. KUPFER

REGULATION OF STEROID HYDROXYLASE GENE EXPRESSION: IMPORTANCE TO

PHYSIOLOGY AND DISEASE

DIANE S. KEENEY a n d MICHAEL R. WATERMAN*

Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, U.S.A.

Abstract--Steroid hydroxylase gene expression is multifactorial in nature, being regulated by tissue-specific, developmental, constitutive and signal transduction systems. The biochemistry of this complex pattern of regulation is not yet clearly elucidated, but studies in several laboratories have led to an understanding of specific aspects of regulation, particularly that involving signal transduction. The complexity of regulation appears to be necessary for normal human physiology because of the wide variety of steroid hormones produced by these enzymes. Genetic diseases associated with the steroid hydroxylases provide examples of how aberrant physiology can result from alterations in the multifactorial regulation of steroid hydroxylase gene expression.

C O N T E N T S

1. Overview 302 2. Genes Encoding Steroid Hydroxylases and their Regulation 303

2.1. Tissue-specific expression 305 2.2. Developmental expression 305 2.3. Signal transduction pathways 306

2.3.1. CYPI IA 307 2.3.2. CYPI IB 309 2.3.3. CYPI7 309 2.3.4. CYP21 310 2.3.5. CYP19 310 2.3.6. P450-related proteins 311 2.3.7. Summary 311

3. Steroid Hydroxylase Genes and Disease 311 3.1. CYP21 312 3.2. CYPI 1A 312 3.3. CYPI IB 312 3.4. CYPI7 312 3.5. CYPI9 313

4. Conclusions and Future Directions 313 Acknowledgments 313 References 313

*Corresponding author. Abbreviations--ACTH, adrenocorticotropin; CAH, congenital adrenal hyperplasia; CRE, consensus

cAMP-response element; CRS, cAMP-response sequence; CYP, cytochrome P450 gene; DHEA, dehydroepi- androsterone; DOC, deoxycorticosterone; P450~o, aldosterone synthase cytochrome P450; P450~m, aromatase cytochrome P450; P450~, cholesterol side-chain cleavage cytochrome P450; P450np, 1 lfl-hydroxylase cytochrome P450; P45017 ~, i 7~-hydroxylase cytochrome P450; P4502t, 2 l-hydroxylas¢ cytochrome P450; SF I, steroidogenic factor 1.

301

302 D. S. KEENEY and M. R. WATERMAN

1. OVERVIEW

In the broadest sense, cytochromes P450 can be classified into two major groups, those that metabolize exogenous compounds and those that metabolize endogenous compounds. Of the latter group, the best studied are the steroid hydroxylases involved in the biosynthesis of steroid hormones (Waterman and Simpson, 1989). Steroid hydroxylases are localized in a variety of steroidogenic cells including adrenocortical cells in the zona glomerulosa, fasciculata and reticularis; testicular Leydig cells; ovarian granulosa, thecal and luteal cells and placental cells. While not always considered to be a steroidogenic tissue, steroid hydroxylases are also localized in various sites of the brain. Steroidogenic tissues produce different steroid hormones depending on their resident composition of steroid hydroxylases. The adrenal cortex has the broadest spectrum of steroid hydroxylases enabling the production of mineralocorticoids, glucocorticoids and androgens within a single gland (Fig. 1). The gonads and placenta are involved primarily in production of sex steroid hormones including estrogens, progestins and androgens. The brain is involved in production of neurosteroids (Jung-Testas et al., 1989), many of which remain to be identified with respect to their structure and function.

Steroid hormone biosynthesis involves cytochromes P450 localized in both the endoplasmic reticulum and mitochondria and the passive diffusion of steroid intermediates between these two subcellular compartments. The primary steroidogenic enzyme localized in virtually all steroidogenic cells is cholesterol side-chain cleavage cytochrome P450 (P450,). This enzyme is localized in the inner mitochondrial membrane and requires an electron-transport chain resident in the mitochondrial matrix for its function (Simpson, 1979). Reducing equivalents from NADPH are transferred to the flavoprotein adrenodoxin reductase and then to the iron-sulfur ‘protein adrenodoxin. Adrenodoxin transfers electrons ultimately to mitochondrial P45Os including P450,, which converts cholesterol to pregnenolone in a three-step process involving hydroxylation at C20 and C22 followed by cleavage of the C-C bond yielding the Cr, steroid pregnenolone. Subsequent steps in steroid hormone biosynthesis take place in the endoplasmic reticulum, and these imprint the unique steroid hormone profile characteristic of different steroidogenic tissues. For example,

P45Q aldo

I P450 aldo

MlTOCHONORlA

AIansfmEDK)M

1 P4SOCZl

P45011@ J

P4JOC21

< &E 11-

ENOOPLASMIC RETICULUM

FIG. 1. Steroidogenic pathways in the adrenal cortex of most species. P450,, cholesterol sidechain cleavage cytochrome P450; P450,,,, 17~1 -hydroxylase cytochrome P450; 3flHSD, 38 -hydroxysteroid dehydrogenase/A’“’ isomerase; P450,,, , 21-hydroxylase cytochrome P450; P450,,,, 1 l/3-hydroxylase cytochrome P450; P450,N,, aldosterone synthase cytochrome P450.

Modified from Zanger et al. (1992).

Steroid hydroxylase gene expression 303

progesterone is a final product of the steroidogenic pathway in the corpus luteum and placenta. In this case, pregnenolone produced by mitochondrial P450~ is converted to progesterone by oxidation of the C-3 hydroxyl. This reaction is catalyzed by a non-P450 enzyme, 3//-hydroxysteroid dehydrogenase/A 5~4 isomerase, in the endoplasmic reticulum. In the production of many other steroid hormones, including the glucocorticoids, androgens and estrogens, pregnenolone is a substrate for microsomal 170t-hydroxylase cytochrome P450 (P450~7,). P45017~ is very unique in that it has two distinct activities, 17~t-hydroxylase and 17,20 lyase, making possible two sequential enzymatic reactions. Products of these reactions, 17a-hydroxypregnenolone and dehydroepi- androsterone (DHEA), are key precursors for different steroidogenic pathways (Kiihn-Velten et al., 1991). This contrasts with P450~, which, as noted above, catalyzes the conversion of cholesterol to pregnenolone in a three-step concerted process with little or no accumulation of steroid intermediates. Like all P450s localized in the endoplasmic reticulum, P450~7~ receives reducing equivalents from NADPH via the ubiquitous microsomal flavoprotein NADPH-cytochrome P450 reductase (Hiwatashi and Ichikawa, 1979). The gonads and adrenal gland are tissues in which P450~7~ provides precursors for different steroidogenic pathways. In the gonads, the two sequential reactions catalyzed by P450~7~ must take place for production of sex steroid hormones. In the latter reaction involving 17,20 lyase activity, the C21 steroid 170t-hydroxypregnenolone is converted to the C~9 androgen DHEA. In the testis, DHEA is converted to testosterone and 50t-dihydrotestosterone by a series of non-P450 enzymatic reactions, whereas in the ovary, it may be converted to estrogen by an additional P450-mediated step catalyzed by aromatase cytochrome P450 (P450 .... ). In contrast to the gonads, P450~7~ in the adrenal gland provides the steroid precursor for another steroidogenic pathway leading to production of glucocorticoids (e.g. cortisol). In this case, 17~t-hydroxypregnenolone produced by the first catalytic reaction of P45017~ is swept through a pathway involving 3fl-hydroxysteroid dehydrogenase/A 5~4 isomerase and a second microsomal cytochrome P450, the 21-hydroxylase cytochrome P450 (P45020, yielding 11-deoxycortisol. 11-De- oxycortisol then finds its way back to the mitoehondrion where 1 l~-hydroxylase cytochrome P450 (P450.a) catalyzes the production of cortisol. In tissues that lack P450,7~, such as adrenocortical cells in the zona glomerulosa or the zona fasciculata/reticularis of certain species (e.g. rat), progesterone is converted to deoxycorticosterone (DOC) by P4502~, which is then converted to corticosterone (also a potent glucocorticoid) by P450Hp. In addition to 1 lfl-hydroxylase activity, zona glomerulosa cells have aldosterone synthase activity. This activity is attributable to a unique mitochondrial cytochrome P450, aldosterone synthase eytochrome P450 (P450,1do), which converts corticosterone to aldosterone, the major mineraloeorticoid. In the rat, both P450~do and P450,~p convert DOC to corticosterone, and both enzymes seem to be present in the zona glomerulosa (Ogishima et al., 1989). Thus, the origin of corticosterone for aldosterone production is unclear. In the mouse, it appears that P450~do predominates in the zona glomerulosa (Domalik et al., 1991). P450~ldo and P450.a are very similar to one another, and only recently has it become evident that these are, in fact, two distinct enzymes (Ogishima et al., 1989).

The expression of genes encoding these steroid hydroxylases involves tissue-specific, developmental and peptide hormone-regulated mechanisms; however, details of these multifactorial regulatory processes are not yet clear for any one steroid hydroxylase gene. In this article, we will provide a progress report as to what is known about the various mechanisms controlling steroid hydroxylase gene expression and how they relate to normal and aberrant physiology.

2. GENES ENCODING STEROID HYDROXYLASES AND THEIR REGULATION

From the viewpoint of physiology, one might predict that the steroid hydroxylase genes are very closely related. For example, with respect to tissue specificity, the genes encoding P450tla and P450~1 are uniquely expressed in the adrenal gland, making it the sole source of mineralocorticoids. From the standpoint of developmental regulation, the timely onset of expression of the genes encoding P450,~c and P450~7~ in the fetus is critical for testosterone production leading to development of the male phenotype. In the adrenal gland, the peptide hormone adrenocorticotropin (ACTH) coordinately enhances the expression of genes encoding P450~, P450tT,, P45021 and P450,p, thereby maintaining optimal steroidogenic capacity in this gland (John et al., 1986). It will be seen that

304 D.S. KEENEY and M. R. W^~I~M^N

1 2 3 4 5 6 7 8 9 10

m m l t m CYP21

1 2 3 4 5 6 7 8

I - - I I - t - l t -H I CYP17

CYP11 A

2 3 4 5 6 7 8 9

1 2 3 4 5 6 78 9

I - I - I t t t t l -B CYP11 B

1 2 3 4

I I . I . I I I I I _ _ - . . . . . .

5 6 7 8 9 10

, ._ - - -I I I I I I I I 1 Kb

CYP19 ' ,

FIG. 2. Structure of the steroid hydroxylase genes encoding CYP2I, CYPIT, CYPIIA, CYPIIB and CYPI9 in humans. Exons are numbered and depicted by solid boxes. Introns

are depicted by solid lines.

even though these physiological events suggest common features among the steroid hydroxylase genes and their regulatory mechanisms, such is not always the case.

The cytochrome P450 superfamily of genes is large and expanding (Nebert et al., 1991). In humans, at least 10 cytochrome P450 (CYP) gene families are known. Members of these gene families are involved in the metabolism of both exogenous and endogenous substrates. The steroid hydroxylases represent four gene families, and the structure of these genes is presented schematically in Fig. 2. Evolutionarily, the oldest of the steroid hydroxylase genes is CYPI9, which encodes P450 ..... the only known member of this family. The CYPI 1 gene family has evolved somewhat more recently and consists of two subfamilies. CYP11A has a single member, which encodes P450s~. CYP11B has more than one member, and includes the genes encoding P450]]a and P450aldo. From an evolutionary perspective, the most recent of the steroid hydroxylase gene families to appear are CYPI7, which has a single member encoding P45017~, and CYP21, which has two members, in many species one being a pseudogene and one encoding P450~.

The single human CYP11A (P450~) gene contains nine exons (Morohashi et ai., 1987) and is located on chromosome 15 (Chung et al., 1986b). The rat CYPI IA gene structure has also been elucidated (Oonk et al., 1990). Two human CYPllB genes (P450H~ and P450alao) have been characterized. These also contain nine exons (Mornet et al., 1989) and are localized on human chromosome 8 (Chua et al., 1987). Two CypllB genes have been characterized in the mouse (Domalik et al., 1991). The single CYPI7 (P45017~) gene has been characterized in humans (Picado-Leonard and Miller, 1987; Kagimoto et al, 1988b); it contains eight exons located on human chromosome 10 (Matteson et al. 1986). This gene has also been characterized in cows (Bhasker et al., 1989) and mice (Youngblood and Payne, 1992). The CYP21 gene (P45020 contains 10 exons localized on human chromosome 10 (White et al., 1984b) and has been characterized in humans (White et al., 1984b; Higashi et al., 1986), cows (Chung et al., 1986a) and mice (White


et al., 1984a). The CYPI9 (P450arom) gene contains at least 10 exons (Means et al., 1989; Harada et al., 1990; Toda et al., 1990) and is located on human chromosome 15 (Chen et al., 1988).

The regulation of gene expression can involve enhancers and promoters in the 5"-flanking sequences or, in some instances, in intronic sequences of a gene, as well as turnover of RNA and the translated protein product. Virtually all of the work on steroid hydroxylase gene expression has focused on regulatory processes involving the Y-flanking region of the various genes. Investigation of steroid hydroxylase gene expression has been carried out in a number of different laboratories. Each laboratory has had its own agenda, and investigations have focused on different genes in different species including rodents, domestic species and humans. By virtue of this diversity of investigative purposes, our present understanding of steroid hydroxylase gene expression is a composite view rather than a complete understanding of the mechanisms in any one species. Not all of the details of expression in any one gene in a given species will coincide with that in other species.

2.1. TISSUE-SPECIFIC EXPRESSION

The tissue-specific expression of genes encoding steroid hydroxylases has profound physiological implications. For example, expression of P450~, P45017~, P4502m and P4501mo in the inner zones of the adrenal cortex (fasciculata and reticularis) of most species leads to glucocorticoid production, while the absence of P45017~ and the presence of P450amdo in the outer zone of the adrenal cortex (glomerulosa) leads to mineralocorticoid production. In the testis, P450~ and P450mT~ are required for androgen production and are the only steroid hydroxylases expressed at significant levels in Leydig cells. In the ovary, different steroid hydroxylases are expressed in different cell types. In thecal cells, P450~ and P450~7~ are expressed leading to androgen production, while in granulosa cells P450~ and P450~m are expressed leading to estrogen production. P450arom is also expressed in fat cells and in the brain. In rodents, P450~ is expressed in the gonads, but it is absent in the zona fasciculata/reticularis of the adrenal gland, resulting in corticosterone being the major giucocorticoid in these species.

The details of both c/s elements and trans-acting factors essential for tissue-specific expression of the steroid hydroxylase genes are unclear. Recently, however, two groups have found that the CYP11A, CYP11 B 1, CYPI 1 B2, CYP17 and CYP21 genes contain a common DNA sequence that binds a nuclear protein found only in steroidogenic cells. Morohashi and colleagues (1992) have named this factor the Ad4-binding protein, while Parker and colleagues call it steroidogenic factor 1 (SF1) (Lala et al., 1992). It seems almost certain that Ad4-binding protein and SF1 are the same protein. This transcription factor may play key roles in the expression of steroid hydroxylases, but only in steroidogenic cells. However, the basis of the different patterns of steroid hydroxylase gene expression among different steroidogenic cells remains to be determined.

2.2. DEVELOPMENTAL EXPRESSION

Neither the pattern of steroid hydroxylase gene expression during fetal development nor its regulation have been extensively investigated. Obviously, the timely onset of expression of these genes is very important in establishing normal physiology. As pointed out earlier, without the precise timing of the onset of expression of P450~ and P45017~, along with other enzymes involved in androgen biosynthesis, the male phenotype (i.e. secondary sexual characteristics) does not develop. In 46 XY individuals where there is a block in androgen biosynthesis, the female phenotype prevails. While this is a dramatic example, it must be recognized that in a variety of steroidogenic tissues, precise control of expression of the steroid hydroxylase genes is crucial for developmental processes to occur normally. Developmental biology is an exploding area of biological research, and the insights gained daily from the study of systems such as Drosophila melanogaster and Caenorhabditis elegans are profound. These organisms, however, do not produce the pattern of steroid hormones found in mammals, and studies of the developmental aspects of steroid hydroxylase gene expression in mammals have lagged behind. Consequently, the topic is somewhat anecdotal at present. Nevertheless, a survey of these results illustrates that the developmental regulation of steroid hydroxylase gene expression is fascinating. One excellent

306 D.S. KEENEY and M. R. WATERMAN

example is the expression of steroid hydroxylase enzymes in the adrenal gland of domestic species. In cows and sheep, the pattern of secretion of ACTH, the primary trophic hormone regulating adrenal steroid hydroxylases, is bimodal during pregnancy. Levels of ACTH in the fetal circulation are relatively high in early gestation, low or undetectable in midgestation and high again in late gestation (Lund et al., 1988; Tangalakis et al., 1989). In the fetal adrenals, P450~c, P4501~a and P4502t are expressed from very early in gestation to term. P45017~, however, is expressed only in fetal adrenals during early and late gestation and is absent at midgestation. These fetal adrenals, therefore, have the capacity to produce cortisol in early and late gestation, but not during midgestation. This is important since in these species cortisol appears to be involved in triggering parturition, and levels of P45017 ~ may play a role in regulating the onset of parturition. It is interesting that P450~7~ expression is readily detectable in fetal testes at all stages of gestation. In porcine conceptuses, it has recently been shown that the reduction of estrogen synthesis after elongation of the conceptus may be due to reduction in the levels of P45017~ (Conley et al., 1992). Patterns of expression of steroid hydroxylases in fetal testes of rabbits (Anderson and Mendelson, 1988) and fetal adrenals and testes of humans (Voutilainen and Miller, 1986) have also been examined. In these cases, unlike that in domestic species, the regulatory stimulus has not been studied. Thus, although we know the patterns of expression of steroid hydroxylases in a number of different fetal tissues in various species, only in the case of bovine fetal adrenal have the levels of steroid hydroxylases been correlated with levels of the stimulus that enhances signal transduction and ultimately regulates gene expression. An area that has yet to be studied is the initiation of expression of steroid hydroxylase genes during development. At present it is unknown at what stage of development in the early fetus steroid hydroxylase genes are first turned on and, more interestingly, what regulatory factors are responsible for initiating steroid hydroxylase gene expression in fetal tissues. The tools of molecular biology make such a study feasible, but generation of meaningful data will require coordination of the skills of molecular biologists with those of animal scientists.

While perhaps not strictly a developmental problem, certain steroidogenic tissues undergo cyclical differentiation during adulthood. Examples include luteinization, the differentiation of the ovarian follicle into a corpus luteum following ovulation and development of the placenta during pregnancy. It must be emphasized, however, that these processes involve differentiation of cells from one type to another. Recently, the pattern of expression of P450~7~ and P450~c in rat placenta has been examined, revealing differential expression of these enzymes during the progression of pregnancy (Durkee et al., 1992). The process of luteinization enables ovarian cells that produce estrogen prior to ovulation to be recruited for the production of progesterone subsequent to ovulation. A result of this differentiative process is that the expression of P450t7 ~ is shut off and expression of P450~ is enhanced. These events lead to progesterone biosynthesis, a hormone essential for the maintenance of a pregnancy (Rodgers et ai., 1986, 1987). The luteinizing hormone surge at the time of ovulation could be the trophic stimulus responsible for enhanced P450~ expression, but the mechanism whereby P45017 ~ expression is halted is unknown. Finally, it is important not only to consider the onset of expression of steroid hydroxylase genes in a developmental context, but also the decline of steroid hormone biosynthesis associated with ageing. Senescence and the loss of expression of P45017~, which must be related to the reduction in testosterone production associated with ageing, are being studied in cultured adrenal cells by Hornsby and colleagues (1987).

2.3. SIGNAL TRANSDUCTION PATHWAYS

Different steroidogenic organs are targets for different trophic peptide hormones derived from the anterior pituitary gland i.e. adrenocortical cells and ACTH, testicular and ovarian cells and luteinizing hormone and follicle-stimulating hormone. Early studies using hypophysectomized rats brought attention to the role of these peptide hormones in regulating the levels of steroid hydroxylase activities. Purvis and colleagues (1978b) showed that the levels of steroid hydroxylases in the adrenal cortex of rats declined following hypophysectomy and could be restored sub- sequently by administration of exogenous ACTH. A similar phenomenon was observed in the testis where loss of steroid hydroxylase activities following hypophysectomy could be restored to normal


levels by administration of human chorionic gonadotropin (Purvis et al., 1973a). Systematic investigation of the regulation of steroid hydroxylase gene expression by ACTH was carried out initially with primary cultures of bovine adrenal cortical cells using immunological and, sub- sequently, molecular biological techniques (Waterman and Simpson, 1989). These studies revealed a coordinate expression of adrenocortical steroid hydroxylase genes in response to ACTH, and this effect of ACTH required new protein synthesis (John et al., 1986). Further, these studies demonstrated that the action of ACTH was mediated by cAMP, and its effect on the adrenal cortex causing maintenance of steroidogenic activity was primarily at the transcriptional level. Unlike many actions of cAMP on the regulation of mRNA and enzyme levels of other genes, i.e. somatostatin and phosphoenolpyruvate carboxykinase, the action of ACTH/cAMP on steroid hydroxylase gene expression is quite slow (John et al., 1986; Roesler et al., 1988). One can imagine that these aspects of peptide hormone regulation of steroid hydroxylase gene expression will also be true for testis, ovary and placenta, the primary difference between tissues being the nature of the peptide hormone itself.

Unlike the investigation of P450 gene expression by xenobiotic compounds, the action of peptide hormones to regulate steroid hydroxylases is not truly an inductive process, rather, one of maintenance. Thus, peptide hormones act in steroidogenic tissues to maintain optimal steroidogenic capacity rather than to induce the expression of a given steroid hydroxylase in response to a particular challenge. Peptide hormones have two modes of regulation of steroid hormone biosynthesis. One is an acute effect leading to enhanced steroid production by rapid mobilization of cholesterol (obligate precursor) into the steroidogenic pathway (Jefcoate et al., 1987). Another is a trophic (chronic) effect leading to enhanced levels of steroid hydroxylases, which, as indicated above, is exerted at the level of gene transcription. This trophic action of cAMP can be imagined to be one of applying constant pressure on the expression of steroid hydroxylase genes so that optimal steroidogenic capacity is maintained, while the acute action of cAMP is to mobilize the steroid hormone precursor into the steroidogenic pathway, thereby leading to rapid increases in the levels of steroid hormones.

Once the genes encoding steroid hydroxylases were characterized, it was possible to search for specific regulatory elements that might be associated with these genes. When this search was carried out using computers, i.e. examination of upstream sequences for common regulatory sequence elements, it was surprising that extensive sequence homologies were not found among the steroid hydroxylase genes. This was surprising in view of the fact that the expression of these genes in response to cAMP appeared to be coordinated. As will be seen, certain common features associated with transcription have been found among the various steroid hydroxylase genes, but the cAMP-dependent mechanisms seem to be quite unique. From an evolutionary standpoint, this suggests that as these genes evolved they recruited their own unique cAMP responsive systems.

2.3.1. C Y P I I A

To evaluate in biochemical terms the mechanisms by which cAMP regulates expression of steroid hydroxylase genes, it is necessary to identify the DNA elements that impart cAMP-responsiveness to the genes and the proteins that bind to these sequences. The general procedure for identifying cAMP-responsive sequences in genes involves deletion mutagenesis of the Y-flanking sequence of a gene. Generally, a large upstream segment of DNA ( > 2 Kb) from the steroid hydroxylase gene of interest is coupled to a reporter gene (e.g. chloramphenicol acetyltransferase, growth hormone, globin). Deletions of the upstream genomic segments from the Y-end are generated using naturally occurring restriction endonuclease sites. A nested set of reporter gene constructs created in this way is transfected into a cell line of choice (often mouse adrenal tumor Y1 cells), and the ability of cAMP to stimulate expression of the reporter gene is measured. Once a region of DNA containing a cAMP-responsive sequence is located, shorter DNA sequences are generated by a variety of techniques, including the use of additional restriction endonuclease sites and/or polymerase chain reaction to eventually locate the minimal sequence conferring cAMP-responsiveness. Such a sequence is referred to as a cAMP-response sequence (CRS). Proteins that bind to a CRS can be studied qualitatively by gel mobility shiR analysis and quantitatively by purification often using DNA affinity chromatography involving the minimal CRS identified previously. In the case of the

308 D.S. KF..ENEY and M. R. WATERMAN

CYP11A gene, studies have been carried out in four different species: human, bovine, mouse and rat. In the bovine CYPI 1A gene, a single CRS has been located at - 118 to - 8 3 bp (Ahlgren et al., 1990; Momoi et al., 1992). (The numbering of 5'-flanking bp is with respect to the site of initiation of transcription. For example, -118 bp for the bovine CYPI1A gene refers to the nucleotide 118 bp to the Y-side of the site of initiation of transcription.) Although the DNA sequence in this region of the gene is relatively well conserved among the bovine, mouse, rat and human CYPllA genes (Momoi et al., 1992), it has not yet been shown to be involved in cAMP-responsiveness in any species other than the bovine. The CRS of bovine CYP1 IA contains no sequence relatedness to other sequences known to confer cAMP-responsiveness such as the consensus cAMP-response element (CRE) in the somatostatin gene (Roesler et al., 1988). In the human CYP I 1A gene, a CRS has been located much further upstream ( - 1697 to - 1523 bp) than that identified in the bovine (Moore et al., 1990; Inoue et al. 1991). Figure 3 includes a schematic view of putative CRS regulatory elements in CYP11A genes. In the mouse, cAMP-responsiveness in the Y-flanking sequence of Cypl IA cannot be assigned to a single element, but requires a combination of several elements that also play a role in constitutive transcription (Rice et al., 1990). In the rat CYP1 IA gene, cAMP-responsiveness has been localized within 940 bp of the 5'-flanking region (Oonk et al., 1990). In both the human CYP11A and murine Cypl 1A genes, constitutive or basal expression may involve sequence elements at multiple sites, those in the human residing around - 150 bp (Moore et al., 1990; Chung et al., 1989). At this juncture, it is unclear whether CYPI 1A genes in different species actually utilize the same or different regulatory elements for cAMP-responsive and constitutive expression.

The proteins that recognize and bind to sequence elements in CYPI 1A genes have yet to be studied extensively. In the mouse, DNA footprint analysis revealed at least three different proteins that bind within the basal regulatory regions of the Cypl IA gene (Rice et al., 1990). In the cow, the ubiquitous transcription factor Spl binds to the minimal CRS ( - 118 to - 100 bp) and has been proposed to be required for cAMP-responsiveness (Momoi et al., 1992). Spl is not generally

-118 +1 b o v i n e C Y P 1 1 A I ~ I

-100

-1697 +1 h u m a n C Y P 1 1 A ~ ~ '~ , I

-1523

-331 -306 +1 b o v i n e C Y P 1 1 B m ~ ] I

-324 -284 -65 +1

m o u s e C y p 1 1 B 2 ~ I -42

-243 -80 +1 b o v i n e C Y P 1 7 E ~ ~ I

-225 -40

-1L~ +1

h u m a n C Y P 2 1 B ~ I -113

-129 +1 b o v i n e CYP21 I ~ ]

-115

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FIG. 3. Sequence elements in steroid hydroxylase genes conferring cAMP-responsiveness. The solid line represents the 5'-flanking region of the CYP genes. Known cAMP-responsive sequences are depicted by the hatched boxes, and their positions are numbered with reference

to the start site of transcription (+ 1).


found to be associated with transcription of cAMP-responsive genes, and surely in the adrenal, as well as other steroidogenic tissues, Spl is associated with cAMP-independent transcription. If Spl is responsible for cAMP-responsiveness of the bovine CYPI 1A gene, it is likely the result of a unique interaction of Spl with the CRS and perhaps an activator of Spl that is specific to steroidogenic cells (possibly a unique cAMP-dependent protein kinase). Because different investi- gators use different reporter genes, different cell systems for transfection and different methods for transcriptional analysis, it is quite difficult to correlate results obtained with bovine, mouse and human CYPI IA genes at present. At least for the cow, it seems that CYPI 1A gene expression in different steroidogenic tissues will be regulated by an interaction of the minimal CRS ( - 1 1 8 to - 100 bp) identified in this gene and the ubiquitous transcription factor Spl.

In addition to ACTH, factors such as vasoactive intestinal peptide (Trzeciak et al., 1987c) and adrenaline (Ehrhart-Bornstein et al., 1991) have been implicated in the positive regulation of the CYP11A gene via a cAMP-mediated mechanism. A number of other factors potentially regulate CYPl lA gene expression via cAMP-independent mechanisms; these include phorbol esters (Trzeciak et al., 1987a; Moore et al., 1990), angiotensin II (Stirling et al., 1990), calcium (Moore et al., 1990), interferon (Orava et al., 1989), epidermal growth factor (Trzeciak et aL, 1987b) and insulin-like growth factor (Veldhuis et al., 1986). While much of the work to localize CRS elements in the various CYP11A genes has been carried out in cultured mouse adrenal cells, it is very clear that CYPI 1A gene expression in ovarian luteal (Lauber et al., 1991) and follicular (Voutilainen et al., 1986; Goldfing et al., 1987; McAllister et al., 1989, 1990; McMasters et al., 1987) cells and testicular Leydig cells (Mason et al., 1984; Mellon and Vaisse, 1989; Hales et al., 1990) is also cAMP-responsive.

2.3.2. C Y P I I B

One of the most intriguing recent developments in the study of steroidogenic enzymes is the realization that, in at least some species, P450j~a and P450atdo are distinct enzymes demonstrating cell-specific localization. In both the rat and human, these enzymes are clearly established to be two distinct gene products, while in the cow, the issue is less clear. The regulation of expression of the genes encoding P450t~p and P450aldo has not yet been compared in detail. In the mouse, the gene encoding P450atdo utilizes a CRS that includes a CRE-like sequence at - 56 to - 49 bp (Rice et al., 1989). The transcriptional response of this gene to cAMP is relatively rapid, whereas that encoding P450Ha, which lacks a CRE-like element, responds more slowly to stimulation via cAMP (Domalik et al., 1991). By comparison with the mouse, even though the bovine CYPllB gene (encoding P450Hp) has a near consensus CRE (Kirita et al. 1990), two elements farther upstream ( - 306 to - 284 bp and - 331 to - 324 bp) apparently function together to impart cAMP-responsiveness to this gene (Honda et al., 1990). Despite the presence of a near consensus CRE, the transcriptional response of bovine CYP11B to cAMP stimulation is relatively slow (John et al., 1986), its rate of response to cAMP being more like that for mouse P450t ~p which lacks a CRE-like element, than mouse P450~do. which utilizes a CRE-like element within its CRS. Figure 3 depicts schematically our present knowledge of the cAMP-dependent regulatory elements in these two members of the CYP11B family. There are presently no data available that describe the nature of nuclear proteins that bind to the CRE-like elements in the CYP11B subfamily, although it will not be surprising if the CRE-binding protein is involved in transcriptional regulation of the gene encoding P450~do, since this gene appears to utilize a CRE-like element. In addition to cAMP-mediated regulation, salt is known to be a very important regulator of P450~Zo (Lauber et al., 1987; Lauber and Miiller, 1989; Malee and Mellon, 1991). The nature of the gene sequences conferring salt-responsiveness is unknown and represents an interesting and important area for future investigation.

2.3.3. C Y P I 7

Expression of P450~7~ in response to cAMP has been studied in bovine adrenal (Zuber et al., 1986) and ovarian (Rodgers et al., 1986) cells, mouse Leydig cells (Hales et al., 1987) and human fetal adrenal cells (Di Blasio et al., 1987). Figure 3 illustrates the positions of known cAMP-respon-

310 D.S. K~r~nv and M. R. WATERMAN

sive regulatory elements in this gene. The bovine CYPI7 gene contains two CRS elements: CRSI resides at -243 to -225 bp and CRSII resides at - 8 0 to - 4 0 bp (Lund et al., 1990). Neither of these CRS elements exhibit sequence homology to other sequences known to confer cAMP- responsiveness, including CRE. To date, a detailed analysis of cAMP-responsiveness CRS has only been carried out with CRSI. A nuclear protein that binds to CRSI is present in most, if not all, tissues, indicating the lack of cell-specific expression. When transfected and expressed in nonsteroidogenic cells, reporter gene constructs containing CRSI fail to show enhanced transcription in response to cAMP stimulation (Zanger et aL, 1991). This result cannot be explained by an absence of CRSI-binding protein, since gel shift analysis indicates its presence in the transfected cells. Interestingly, the addition of partially purified CRSI-binding protein from steroidogenic cells to an in vitro transcription system prepared from nonsteroidogenic cells resulted in enhanced reporter gene transcription; this result was obtained despite the background level of CRSI-binding protein contributed by the nonsteroidogenic cells. Based on these results, it is postulated that the tissue-specificity associated with cAMP-responsive CYPI 7 gene transcription resides in the ability of steroidogenic cells to activate the CRSI-binding protein. That is, the CRSI-binding protein is expressed ubiquitously, but the system that activates the binding protein (perhaps a unique cAMP-dependent protein kinase) may be expressed only in steroidogenic cells. The characterization of proteins that recognize and bind CRSII is incomplete. It is unknown whether CRSI or CRSII is present in CYPI 7 genes from species other than the bovine, although sequence homology would suggest that perhaps both are present in the human CYP17 gene. In addition to cAMP-mediated regulation of CYP17 gene transcription, glucocorticoids also have been shown to positively regulate the levels of the CYP17 gene product in the ovine placenta (France et al., 1988). The CYP17 gene seems to be quite sensitive to negative regulators as well. For example, in various steroidogenic cells, interferons (Orava et al., 1989), androgens (Hales et al., 1987) and transforming growth factor-fl (Perrin et al., 1990) all have been shown to negatively regulate expression of P45017~, the CYPI7 gene product.

2.3.4. C Y P 2 1

The CYP21 gene family has two members; one encodes a pseudogene and the other a functional gene. In the mouse, Cyp21A is the functional gene and Cyp21B is the pseudogene. In humans, the reverse is true: CYP21A is the pseudogene and CYP21B is the functional gene. The known cAMP regulatory elements in this gene are depicted schematically in Fig. 3. The functional human gene, CYP21 B, contains a single CRS at - 126 to - 113 bp, while the bovine gene contains one at nearly the same position (Kagawa and Waterman, 1990, 1991). Two nuclear proteins bind to the CRS in the human CYP21B gene, the ubiquitous transcription factor Sp I and a protein designated ASP that is found in a limited number of cell types in addition to adrenal cells (Kagawa and Waterman, 1991). This issue is relevant because the CYP21 gene product P450~1 is expressed only in adrenal cells. Within the CRS, the binding regions for ASP and Spl overlap extensively, and the ASP-binding region ( - 126 to - 113 bp) seems to be required for cAMP-responsiveness. Using the ASP-binding region as an affinity site, the ASP protein has been purified and shown to enhance transcription of reporter genes coupled to this CRS. An antibody has been prepared against purified ASP (Kagawa and Waterman, 1992), and we are hopeful that an understanding of its structure and, ultimately, its function are forthcoming. The functional murine gene Cyp21A contains a sequence conferring cAMP-responsiveness within the proximal 330 bp of the Y-flanking region (Handler et aL, 1988). Constitutive expression of CYP21 seems to be stronger than for other steroid hydroxylase genes in the adrenal gland, and may involve multiple binding sites for nuclear proteins.

2.3.5. C Y P I 9

Of all the steroid hydroxylase genes, CYP19 is structurally the most complex (Fig. 2); its regulation appears to be the most complicated as well. Perhaps this is because CYP19 is expressed in steroidogenic (gonads and placenta) as well as nonsteroidogenic (adipose and brain) tissues. Certainly cAMP-dependent transcription is one mechanism controlling the levels of the CYPI9 gene product, P450arom. In adipose stromal (Evans et al., 1986) and ovarian (Steinkampf et al., 1988;


Hickey et aL, 1990) cells, cAMP regulates CYPI9 gene expression, and experiments are now underway to localize key regulatory elements within this gene (Toda et aL, 1990, 1992). An additional level of complexity is associated with the CYPI9 gene as it is now clear that it contains tissue-specific promoters (Mahendroo et al., 1991; Means et al., 1991).

2.3.6. P450-related Proteins

Levels of both adrenodoxin (Okamara et al., 1987) and adrenodoxin reductase (Hanukoglu et al., 1990), which are required for transfer of reducing equivalents from NADPH to mitochondrial P450s (P450~, P450.a and P450a~ao), are known to be enhanced by cAMP. The gene encoding adrenodoxin has been characterized in cows (Sagara et al., 1990) and humans (Chang et al., 1988, 1990), while that encoding adrenodoxin reductase has been characterized in humans (Lin et al., 1990). However, only in the bovine adrenodoxin gene have CRS elements been defined. This gene contains two promoters, one in intron 1 and one in the 5'-flanking sequence of the gene (Kagimoto et al., 1988a). Each promoter has its own associated CRS; neither CRS exhibits sequence homology to other sequences known to confer cAMP-responsiveness (Chen and Waterman, 1992). The promoter within intron 1 (ADXP2) is the stronger of the two, and the CRS associated with it (CRS2) is the most potent CRS. The sequence of CRS2 resembles that of the Spl-binding site, but the nature of the nuclear proteins that recognize CRS2 are unknown. As an aside, these adrenodoxin promoters, which function to regulate gene transcription in steroidogenic tissues, are also functional in nonsteroidogenic tissues that express adrenodoxin, such as kidney for vitamin D metabolism and the liver for bile acid biosynthesis. Whether cAMP is important in regulating adrenodoxin gene transcription in these nonsteroidogenic tissues remains to be determined.

Although the gene encoding the ubiquitous flavoprotein NADPH-cytochrome P450 reductase has been characterized, regulatory elements have not. It has been shown, however, that cAMP enhances P450 reductase levels in primary bovine adrenocortical cells (Dee et al. 1985). Therefore, it will not be surprising if CRS elements are also found to be associated with this gene.

2.3.7. Summary

Given the fact that steroid hydroxylases work in concert for the production of essential and potent steroid hormones, it seems surprising that the regulation of the genes encoding these P450s is so complex. We might not be too surprised at the multifactorial regulation since steroid hydroxylase gene expression requires tissue-specific, developmental, basal (constitutive) and stimulus-dependent transcription, but the fact that cAMP-responsiveness is so complex is quite a surprise and inexplicable. In the adrenal, for example, ACTH coordinately regulates the expression of four steroid hydroxylase genes in order to maintain optimal steroidogenic capacity in this gland. Why then does each gene require its own unique cAMP-responsive system? One might predict that because the CYP11A and CYP11B genes both encode mitochondrial P450s and share the same evolutionary lineage, they would also share common regulatory systems. One might also predict that because the CYP17 and CYP21 both encode microsomal P450s and share an evolutionary lineage that differs from that of the CYPI 1 gene family, these genes might also share a common regulatory system. These predictions prove not to be the case. Rather, the greatest commonality is observed between CYP11A, which encodes the widely distributed mitochondrial steroidogenic enzyme P450~c, and CYP21, which encodes the adrenal-specific microsomal steroidogenic enzyme P45021. The significance of the relatedness of the CRS elements in CYPllA and CYP21, or the general lack of relatedness between the CRS elements of other coordinately regulated steroid hydroxylase genes, is obscure. We can only hope that once the transcription factors that bind to these different CRS elements are characterized, the meaning of this diversity of regulatory systems will become clear.

3. STEROID HYDROXYLASE GENES AND DISEASE

From the discussion of the regulation of steroid hydroxylase gene expression presented above, two things are evident. First, this is a very complex process involving many different levels of

312 D.S. KF~NEY and M. R. WATEgraAN

regulation. Second, we do not yet know a great deal about the biochemistry of these complex regulatory systems. Nevertheless, it is not unreasonable at this juncture to ask what effect aberrant regulation of steroid hydroxylase gene expression would have on normal human physiology. While defects in the regulation of steroid hydroxylase gene expression are unknown, it can be anticipated that this will be found to be a cause of human disease. Yet even now, we have many examples of deficiencies in steroid hydroxylase activities, which provide an excellent view as to what clinical phenotypes can be expected from reduced expression of any one of the steroid hydroxylase genes. These genetic diseases are known collectively as congenital adrenal hyperplasia (CAH). Most of the cases of CAH that have been studied at the molecular level result from mutations within the coding sequences of the CYP genes. The most common of these diseases are those associated with 21-hydroxylase deficiency. The reason that this genetic disease is much more common than other potential causes of CAH is because the CYP21 gene family involves two closely linked members that reside within the human lymphocyte antigen major histocompatibility complex.

3.1. CYP21

Greater than 90% of the known cases of CAH are examples of P4502~ deficiency. This gene and its pseudogene are closely linked with the human lymphocyte antigen locus. The pseudogene contains an 8-bp deletion in exon 3, a single bp insertion in exon 7, and a single base change leading to a stop codon in exon 8. Most of the known causes of 21-hydroxylase deficiency are due to gene conversions between the functional gene and the pseudogene, and in some cases gene deletion is involved. Point mutations have also been found in a few cases. The clinical features and molecular genetics of this form of CAH are summarized in an excellent review by Morel and Miller (1991). Complete deficiency of P4502~ leads to the absence of aldosterone, which leads to the salt-wasting form of this disease due to Na ÷ loss and K ÷ and H ÷ retention; death ensues without treatment. In addition, the accumulation of non-21-hydroxylated steroids leads to excess androgen production and virilization of the external genitalia. This is a relatively common genetic disease, and presumably examples of this type of CAH resulting from altered levels of gene expression will be found to be clinically similar to the known examples of gene deletion.

3.2. CYP11A

Deficiency in P450~ activities will lead to absence of production of all classes of steroid hormones. Thus, one may expect affected individuals to exhibit hypertension, a female phenotype, and undetectable levels of glucocorticoids in the peripheral circulation. A few such cases have been reported in the literature. Steroid hormone replacement therapy, begun at an early age, should lead to a relatively normal physiology in these individuals. In three individuals where CYPI1A deficiency has been suspected, the coding sequence of this gene has been examined. In none of these cases were mutations observed (Lin et al., 1991). Therefore, if the disease in these individuals has been diagnosed correctly, mutations in the regulatory region or introns may be the cause.

3.3. CYPI IB

Symptoms associated with 1 lfl-hydroxylase deficiency include hypertension and virilization. In one case the molecular basis of 11 fl-hydroxylase deficiency has been found to be due to a single amino acid change (White et al., 1991). The discovery that P450aldo is distinct from P450)~ B helps explain the rather varied clinical profile associated with this deficiency. We can expect mutations to be found in one or, perhaps in some cases, both of the genes in this gene subfamily.

3.4. CYP17

Absence of P450~7~ leads to absence of cortisol and androgen production and quite often hypertension due to elevated levels of DOC and corticosterone. While the salt balance and glucocorticoid levels in these individuals can be controlled, the phenotypic result of the inability to produce sex steroid hormones cannot be corrected. Thus, effects on sexual development in these individuals are irreversible. More than 125 cases of this type of CAH have been reported in the


literature (Yanase et al., 1991), and in more than 20 of these cases the molecular basis oftbe disease has been determined. In all of the cases studied, a defect has been found in the coding sequence resulting from a change, insertion or deletion of an amino acid or, alternatively, an insertion or deletion of nucleotides that alters the reading frame of the gene. In all cases studied so far, the molecular basis of the defect explains the clinical symptoms. The number of cases of P450~ deficiency reported and the variety of clinical symptoms associated with these deficiencies suggests that mutations in the regulatory regions of CYP17 will also be found.

3.5. CYPl9

Little is known about aromatase deficiency. Recently the molecular basis of an example of partial aromatase deficiency has been reported (Harada et al., 1992).

4. CONCLUSIONS AND FUTURE DIRECTIONS

The elucidation of the biochemical events associated with regulation of steroid hydroxylase gene expression is rapidly emerging. By the end of this century, we will have a detailed understanding of the tissue-specific, developmental and signal transduction pathways leading to the diversity of expression of these genes. Understanding of the multifactorial nature of their expression will contribute in the broad sense to our general understanding of gene expression, as well as to that of the specific genes themselves. With respect to human disease associated with these genes, we can also expect to learn more during this same time period. We do not, however, forsee this as an area where genetic screening or gene therapy will become major efforts around the world, because CAH is generally non-life threatening and, in those cases where it is, it can be treated relatively easily with steroid replacement therapy. Furthermore, the solution of the molecular basis of these defects will probably not be an endless investigative effort--how many different examples of CAH should be evaluated in detail? Certainly, application of these gene mutations to structure/function analyses of P450s will be useful once crystal structures for the steroid hydroxylases become available.

Perhaps the most important information that will be forthcoming from studies of steroid hydroxylase genes and their regulatory mechanisms will be evolutionary. The SFl/Ad4-binding protein is found to be related to a transcription factorfushi tarazu-factor 1 from D. melanogaster. An understanding of this and other such relationships associated with steroid hydroxylases should provide new insight into the evolution of human physiology.

Acknowledgements--M.R.W. is supported in part by USPHS grant DK28350. The editorial assistance of Madene Jayne is greatly appreciated.

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MEANS, G. D., MAHENDROO, M. S., CORBIN, C. J., MATHIS, J. M., POWELL, F. E., MENDELSON, C. R. and SIMPSON, E. R. (1989) Structural analysis of the gene encoding human aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. J. biol. Chem. 264: 19385-19391.

JPT 58/3--E

316 D.S . KEENEY and M. R. WATERMAN

MEANS, G. D., KILGORE, M. W., MAHENDROO, M. S., MENDELSON, C. R. and S1MPSON, E. R. (1991) Tissue- specific promoters regulate aromatase cytochrome 1)450 gene expression in human ovary and fetal tissues. Molec. Endocr. 5: 2005-2013.

MELLON, S. H. and VAISSE, C. (1989) cAMP regulates P450~ gene expression by a cycloheximide-insensitive mechanism in cultured mouse Leydig MA-10 cells. Proc. natn. Acad. Sci. U.S.A. 86: 7775-7779.

MOMOI, K., WATERMAN, M. S., SIMPSON, E. R. and ZANGER, U. M. (1992) 3',5'-Cyclic adenosine monophosphate-dependent transcription of the CYPI IA (cholesterol side chain cleavage cytochrome P450) gene involves a DNA response element containing a putative binding site for transcription factor Spl. Molec. Endocr. 6: 1682-1690.

MOORE, C. C. D., BRENTANO, S. T. and MILLER, W. L. (1990) Human P450~ gene transcription is induced by cyclic AMP and repressed by 12-O-tetradecanoylphorboi-13-acetate and A23187 through independent cis elements. Molec. cell. Biol. 10: 6013-6023.

MOREL, Y. and MILLER, W. L. (1991) Clinical and molecular genetics of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. In: Advances in Human Genetics, Vol. 20, pp. 1-68, HARRIS, H. and HIRSCHHORN, K. (¢ds) Plenum Press, New York.

MORNET, E., DUPONT, J., VITEK, A. and WHITE, P. C. (1989) Characterization of two genes encoding human steroid 1 l~-hydroxylase (P45011B). J. biol. Chem. 264: 20961-20967.

MOROHASHI, K., SOGAWA, K., OMURA, T. and FUJII-KURIYAMA, Y. (1987) Gene structure of human cytochrome P450(scc) cholesterol desmolase. J. Biochem. 101: 879-887.

MOROHASHI, K., HONDA, S., INOMATA, Y., HANDA, H. and OMURA, T. (1992) A common trans-acting factor, Ad4-binding protein, to the promoters of steroidogenie P450s. J. biol. Chem. 267: 17913-17919.

NEBERT, D. W., NELSON, D. R., COON, M. J., ESTABROOK, R. W., FEYEREISEN, R., FUJII-KUR1YAMA, Y., GONZALEZ, F. J., GUENGERICH, F. P., GUNSALUS, I. C., JOHNSON, E. F., LOPER, J. C., SATO, R., WATERMAN, M. R. and WAXMAN, D. J. (1991) The P450 superfamily: update on new sequences, gene mapping, and recommended nomenclature. DNA Cell Biol. 10: 1-14.

OGISHIMA, T., MITANI, F. and ISHIMURA, Y. (1989) Isolation of aldosterone synthase cytochrome P450 from zona glomerulosa mitochondria of rat adrenal cortex. J. biol. Chert:. 264: 10935-10938.

OKAMURA, T., KAGIMOTO, M., SIMPSON, E. R. and WATERMAN, M. R. (1987) Multiple species of bovine adrenodoxin mRNA: occurrence of two different mitochondrial precursor sequences associated with the same mature sequence. J. biol. Chem. 262: 10335-10338.

OONK, R. B., PARKER, K. L., GmSON, J. L. and RICHARDS, J. S. (1990) Rat cholesterol side-chain cleavage cytochrome P450 (P450scc) gene: structure and regulation by cAMP in vitro. J. biol. Chem. 265: 22392-22401.

ORAVA, M., VOUTILAINEN, R. and VIHKO, R. (1989) Interferon-~, inhibits steroidogenesis and accumulation of mRNA of the steroidogenic enzymes P450~¢c and P450c~7 in cultured porcine Leydig cells. Molec. Endocr. 3: 887-894.

PERRIN, A., PASCAL, O., DEFAYE, G., FEIGE, J.-J. and CHAMBAZ, E. M. (1990) Transforming growth factor fl~ is a negative regulator of steroid 17ct-hydroxylase expression in bovine adrenocortical cells. Endocrinology 128: 357-362.

PICADO-LEONARD, J. and MILLER, W. L. (1987) Cloning and sequence of the human gene for P450c17 (steroid 17~-hydroxylase/17,20 lyase): similarity with the gene for P450c21. DNA 6: 439-448.

Punvis, J. L., CANICK, J. A., LATIF, S. A., ROSENBAUM, J. H., HOLOGGITAS, J. and MENARD, R. H. (1973a) Lifetime of microsomal cytochrome P450 and steroidogenic enzymes in rat testis as influenced by human chorionic gonadotropin. Arch. Biochem. Biophys. 159: 39-49.

PURVIS, J. L., CANICK, J. A., MASON, J. I., ESTAnROOK, R. W. and MCCART.Y, J. L. (1973b) Lifetime of adrenal cytochrome 1)450 as influenced by ACTH. Ann. N Y Acad. Sci. 212: 319-342.

RICE, D. A., A1TKEN, L. D., VANDENBARK, G. R., Mouw, A. R., FRANKLIN, A., SCHIMMER, B. P. and PARKER, K. L. (1989) A cAMP-responsive element regulates expression of the mouse steroid 1 lfl-hydroxylase gene. J. biol. Chem. 264:14011-14015.

RICE, D. A., KIRKMAN, M. S., AITKEN, L. D., MOUW, A. R., SCHIMMER, B. P. and PARKER, K. L. (1990) Analysis of the promoter region of the gene encoding mouse cholesterol side chain cleavage enzyme. J. biol. Chem. 265: 11713-11720.

RODGERS, R. J., WATERMAN, M. R. and SIMPSON, E. R. (1986) Cytochromes P450=c, P450~7,, adrenodoxin and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P450 reductase in bovine follicles and corpora lureR. Changes in specific contents during the ovarian cycle. Endocrinology 118: 1366-1374.

RODGERS, R. J., WATERMAN, M. R. and SIMPSON, E. R. (1987) Levels of messenger ribonucleic acid encoding cholesterol side chain cleavage cytochrome P450, 17g-hydroxylase cytochrome P450, adrenodoxin, and low density lipoprotein receptor in bovine follicles and corpora lutea throughout the ovarian cycle. Molec. Endocr. 1: 274-279.

ROESLER, W. J., VANDENBARK, G. R. and HANSON, R. W. (1988) Cyclic AMP and the induction of eucaryotic gene transcription. J. biol. Chem. 263: 9063-9066.

SAGARA, Y., SAWAE, H., KIMURA, A., SAGARA-NAKANO, Y., MOROHASHI, K., MIYOSHI, K. and HORIUCHI, T. (1990) Structural organization of the bovine adrenodoxin gen¢. J. Biochem. 107: 77-83.

SIMPSON, E. R. (1979) Cholesterol side-chain cleavage cytochrome I)450 and the control of steroidogenesis. Molec. Cell. Endocr, 13: 213-227.

STEINKAMPF, M. P., MENDELSON, C. R. and SIMPSON, E. R. (1988) Effects of epidermal growth factor and


insulin-like growth factor I on the levels of mRNA encoding aromatase cytochrome P--450 of human ovarian granulosa cells. Molec. cell. Endocr. 59: 93-99.

STIRLING, D., MAGNESS, R. R., STONE, R., WATERMAN, M. R. and SIMPSON, E. R. (1990) Angiotensin II inhibits luteinizing hormone-stimulated cholesterol side chain cleavage expression and stimulates basic fibroblast growth factor expression in bovine luteal cells in primary culture. J. biol. Chem. 265: 5-8.

TANGALAKIS, K., COGHLAN, J. P., CONNELL, J., CRAWFORD, R., DARLING, P., HAMMOND, V. E., HARALAMBIDIS, J., PENSCHOW, J. and WINTOUR, E. M. (1989) Tissue distribution and levels of gene expression of three steroid hydroxylases in ovine fetal adrenal glands. Acta Endocr. 120: 225-232.

TODA, K., TERASHIMA, M., KAWAMOTO, T., SUMIMOTO, H., YOKOYAMA, Y., KURIBAYASHI, I., MITSUUCHI, Y., MAEDA, T., YAMAMOTO, Y., SAGARA, Y., IKEDA, H. and SHIZUTA, Y. (1990) Structural and functional characterization of human aromatase P450 gene. Eur. J. Biochem. 193: 559-565.

TODA, K., MIYAHARA, K., KAWAMOTO, T., IKEDA, H., SAGARA, Y. and SHIZUTA, Y. (1992) Characterization of a cis-acting regulatory element involved in human aromatase P450 gene expression. Fur. J. Biochem. 205: 303-309.

TRZECIAK, W. H., DUDA, T., WATERMAN, M. R. and SIMPSON, E. R. (1987a) Tetradecanoyl phorbol acetate supresses follicle-stimulating hormone induced synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells. J. biol. Chem. 262: 15246-15250.

TRZECIAK, W. H., DUDA, T., WATERMAN, M. R. and SIMPSON, E. R. (1987b) Effects of epidermal growth factor on the synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells in primary culture. Molec. cell. Endocr. 52: 43-50.

TRZECIAK, W. H., WATERMAN, M. R., SIMPSON, E. R. and OJEDA, S. R. (1987c) Vasoactive intestinal peptide regulates cholesterol side chain cleavage cytochrome P450 (P450~) gene expression in granulosa cells from immature rat ovaries. Molec. Endocr. 1: 500-504.

VELDHUIS, J. D., RODGERS, R. J., DEE, A. and SIMPSON, E. R. (1986) The insulin-like growth factor, so- matomedin C, induces the synthesis of cholesterol side-chain cleavage cytochrome P450 and adrenodoxin in ovarian cells. J. biol. Chem. 261: 2499-2502.

VOUTILAINEN, R. and MILLER, W. L. (1986) Developmental expression of genes for the steroidogenic enzymes P450~ (20,20-desmolase), P450c]7 (l 7ct-hydroxylase/17,20-1yase), and P450c21 (2 l-hydroxylase) in the human fetus. J. clin. Endocr. Metab. 63: 1145-1150.

VOUTILAINEN, R., TAPANAINEN, J., CHUNG, B.-C., MATTESON, K. J. and MILLER, W. L. (1986) Hormonal regulation of P450~ (20,22-desmolase) and P450t7 ~ (17g-hydroxylase/17,20 lyase) in cultured human granulosa cells. J. din. Endocr. Metab. 63: 202-207.

WATERMAN, M. R. and SIMPSON, E. R. (1989) Regulation of steroid hydroxylase gene expression is multifactorial in nature. Rec. Prog. Horm. Res. 45: 533-566.

WHITE, P. C., CHAPLIN, D. D., WEIS, J. H., DUPONT, B., NEW, M. I. and SEIDMAN, J. G. (1984a) Two steroid hydroxylase genes are located in the murine S region. Nature 312: 465-467.

WHITE, P. C., NEW, M. I. and DUPONT B. (1984b) HLA-linked congenital adrenal hyperplasia results from a defective gene encoding a cytochrome P450 specific for steroid 21-hydroxylation. Proc. HatH. Acad. Sci. U.S.A. 81: 7505-7509.

WHITE, P. C., DUPONT, J., NEW, M. I., LEIBERMAN, E., HOCHBERG, Z. and ROSLER, A. (1991) A mutation in CYP11B1 (Arg 448~His) associated with steroid 1 lfl-hydroxylase deficiency in Jews of Moroccan origin. J. clin. Invest. 87: 1664-1667.

YANASE, T., SIMPSON, E. R. and WATERMAN, M. R. (1991 ) i 7~-Hydroxylase/17,20-1yase deficiency: from clinical investigation to molecular definition. Endocr. Rev. 12: 91-108.

YOUNGBLOOD, G. L. and PAYNE, A. H. (1992) Isolation and characterization of the mouse P450 17~t-hydroxylase/Cl7-20 lyase gene (Cypl7): transcriptional regulation of the gene by cyclic adenosine Y,5'-monophosphate in MA-10 Leydig cells. Molec. Endocr. 6: 927-934.

ZANGER, U. M., LUND, J., SIMPSON, E. R. and WATERMAN, M. R. (1991) Activation of transcription in cell-free extracts by a novel cAMP responsive sequence from the bovine CYPI7 gene. J. biol. Chem. 266: 1141%11420.

ZANGER, U. M., KAGAWA, N., LUND, J. and WATERMAN, M. R. (1992) Distinct biochemical mechanisms for cAMP-dependent transcription of CYPI7 and CYP21. FASEB J. 6: 719-723.

ZUBER, M. X., JOHN, M. E., OKAMURA, T., SIMPSON, E. R. and WATERMAN, M. R. (1986) Bovine adrenocortical cytochrome P-450~7~. Regulation of gene expression by ACTH and elucidation of primary sequence. J. biol. Chem. 261: 2475-2482.

regulation of steroid hydroxylase gene expression: importance to physiology and disease

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