Regulation of Proteins in the Cholesterol Side-Chain Cleavage System in JEG-3 and Y-1 Cells*
STEPHEN M. BLACK, GRAZYNA D. SZKLARZ, JENNIFER A. HARIKRISHNA, DONG LIN, C. ROLAND WOLF, AND WALTER L. MILLER
Department of Pediatrics and the Metabolic Research Unit, University of California, San Francisco, California 94143-0978; and Imperial Cancer Research Fund (C.R.W.), University of Edinburgh, Edinburgh, United Kingdom
ABSTRACT
The conversion of cholesterol to pregnenolone, the rate-limiting step in steroid hormone synthesis, occurs on mitochondrial cytochrome P450scc, which catalyzes this reaction by receiving electrons from NADPH via a flavoprotein [adrenodoxin reductase (AdRed)] and an iron sulfur protein [adrenodoxin (Adx)]. The behavior of the genes and mRNAs encoding these proteins has been studied in several systems, but little is known about the behavior of the human proteins. Using cloned cDNAs for human P450scc and AdRed, we constructed bacterial expression vectors to make milligram quantities of the corresponding proteins. These, plus purified human Adx similarly prepared by Dr. L. Vickery, were injected into rabbits to raise antiserum to each of the proteins. Each antiserum was highly specific and did not cross-react with other mitochondrial proteins detectable by Western blotting. Human JEG-3 choriocarcinoma cells and mouse Y-1 adrenocortical carcinoma cells were then incubated for 0-24 h with 1 mM 8-bromo- cAMP (8Br-cAMP) or 30 nM phorbol 12-myristate 13-acetate (PMA;
phorbol ester) plus 1 uM A23187 (calcium ionophore) to activate the protein kinase-A and -C pathways, respectively. In JEG-3 cells, 8Br- cAMP increased and PMA/A23187 slightly decreased the abundance of P450scc and Adx, but neither treatment had a detectable effect on AdRed. The production of pregnenolone by these cells increased 3-fold in response to 8Br-cAMP and fell to one third in response to PMA/ A23187. In Y-1 cells, 8Br-cAMP increased the abundance of all three proteins, while PMA/A23187 decreased the abundance of P450scc and Adx. The production of pregnenolone by these cells increased 9-fold in response to 8Br-cAMP and was unaffected by TPA/A23187. These studies show that the three proteins of the cholesterol side-chain cleavage system behave in response to 8Br-cAMP and PMA/A23187 as predicted from the study of their genes and mRNAs, indicating that the chronic regulation of steoridogenesis in these cell systems is regu- lated principally at the level of mRNA abundance. (Endocrinology 132: 539-545, 1993)
T HE FIRST and rate-limiting step in steroid hormone synthesis is the conversion of cholesterol to pregneno- lone by the cholesterol side-chain cleavage system, which consists of three mitochondrial components (for review, see Ref. 1). A flavoprotein, commonly termed adrenodoxin re- ductase (AdRed), receives electrons from NADPH, then transfers them to an iron sulfur protein, generally termed adrenodoxin (Adx), which subsequently passes them to the cholesterol side-chain cleavage enzyme, P450scc. One pair of electrons must pass through this system for each of the three sequential reactions, 20@-hydroxylation, 22-hydroxyl- ation, and C20,22 bond scission, to convert cholesterol to pregnenolone. AdRed and Adx are loosely adherent to the inner mitochondrial membrane (2), while P450scc is tightly bound. The human nuclear genes for each of these compo- nents have been cloned (3-5), and their chromosomal loca- tions determined (6). The human cDNAs for each have also been cloned (7-9), showing that the corresponding mRNAs encode proteins with typical mitochondrial leader sequences. Adx and AdRed serve as generic electron transport proteins for all mitochondrial forms of cytochrome P450, and hence, their mRNAs are found in all tissues examined (8, 10), but
Received August 3, 1992.
Address all correspondence and requests for reprints to: Walter L. Miller, M.D., Building MR-IV, Room 209, University of California, San Francisco, California 94143-0978.
detectable amounts of P450scc mRNA are found only in the classical steroidogenic tissues, adrenal, testis, ovary, and pla- centa (7, 11), in a developmental pattern of expression (12, 13).
Steroidogenesis is regulated by ACTH in the adrenal and by gonadotropins in the ovary, testis, and placenta, all acting through cAMP and protein kinase-A as the intracellular second messenger system (for reviews, see Refs. 1, 14, 15). In the adrenal zona glomerulosa, mineralocorticoid synthesis is regulated primarily by angiotensin-II, which acts through intracellular Ca2+ and protein kinase-C (16). Both of these systems elicit a rapid induction of steroidogenesis, apparently by increasing the flux of free cholesterol into mitochondria. Long term exposure to ACTH or gonadotropins will chroni- cally stimulate steroidogenesis through cAMP’s action to stimulate the accumulation of the mRNAs for the various human steroidogenic enzymes (11, 17-20), principally as a result of increased gene transcription (for reviews, see Refs. 21 and 22). By contrast, long term exposure to agonists of the protein kinase-C system either have no effect or suppress the transcription of genes encoding steroidogenic enzymes (23-25).
While the accumulation of mRNAs for human steroido- genic enzymes and transcription of the corresponding genes have been studied extensively (reviewed in Refs. 14, 21, and 22), little is known about the abundance of the human enzymes themselves. The abundance of steroidogenic en- zymes has been studied in primary cultures of bovine adre-
nocortical cells (15), but corresponding studies in human tissues have been limited by the lack of antibodies to the human enzymes. We now report the production of milligram amounts of human steroidogenic enzymes in bacteria and the use of these recombinant proteins to raise specific anti- sera. These antisera permitted characterization of the patterns of induction of P450scc, Adx, and AdRed in human JEG-3 cells and mouse adrenocortical Y-1 cells treated with 8- bromo-cAMP (8Br-cAMP) to activate the PKA system or phorbol ester plus calcium ionophore to activate the PKC system.
Materials and Methods
Plasmid construction
E. coli strains HMS174 and BL21(DE3)plysS and the cloning vectors pET-3b and pET-3c were obtained from Dr. W. Studier (Brookhaven National Laboratory, Long Island, NY). The pET(a-c) class of expression vectors (26) has a unique BamHI restriction site in each of the three possible coding frames in gene 10 of phage T7, allowing any foreign cDNA to be expressed. To construct the P450scc expression vector, the human P450scc cDNA in pUC18 (7) was digested with EcoRI and Rsal to produce a truncated P450scc cDNA lacking sequences encoding the mitochondrial leader sequence and the first 23 amino acids of the mature protein. The cDNA was isolated using Geneclean II (Bio 101, La Jolla, CA) and treated with Klenow polymerase to render the DNA
blunt-ended. This was then ligated to pET-3b, which had been digested with BamHI and treated with Klenow polymerase, to yield the plasmid pET/P450scc (Fig. 1A). To construct a vector expressing human AdRed in E. coli, the 600-basepair EcoRI fragment containing the translational terminator sequence was first deleted from the bacterial expression vector pET-3c. A 1.7-kilobase BglII-EcoRI fragment derived from the full-length human cDNA (9) contained all of the coding region of mature AdRed except the first eight amino acid residues and was cloned into the vector at BamHI and EcoRI sites. The translational terminator se- quence was then replaced at the EcoRI site to yield expression vector pET/AdRed (Fig. 1B).
Protein expression and gel electrophoresis
E. coli strain BL21(DE3)plysS containing either pET/P450scc or pET/ AdRed was grown in L-broth to an OD600 of 0.3, induced with 0.4 mm isopropyl 8-D-thiogalactoside (IPTG), and harvested 3 h later. The cell pellet was resuspended in 50 mm Tris-HCI (pH 7.5)-0.1 mm EDTA and lysed by freeze-thawing in dry ice. MgCl2 was added to a final concen- tration of 5 mm, and the culture was incubated at room temperature with 400 U Q1 DNase until the viscosity was reduced. The resulting supernatant was then diluted 1:1 with 2 x loading dye [2% sodium dodecyl sulfate (SDS)-5% 8-mercaptoethanol, 10% glycerol, and 0.005% bromphenol blue in 0.05 M Tris, pH 6.8] and electrophoresed through a SDS-9% polyacrylamide gel. The gel was rinsed twice in distilled water, then the proteins were made visible with ice-cold 250 mm KCI-1 mm dithiothreitol, and the gel was washed again in ice-cold 1 mm dithio- threitol (27). The band corresponding to the expressed protein was excised and minced by passage through an 18-gauge needle. The result- ing protein-acrylamide slurry was lyophilized and used for immuniza-
A
B
EcoRI
EcoRI
BamHI
EcoRI
Rsal
EcoRI
BamHI
Tr
Pr
Bgill
Tr
Bgill
pUC/AdRed
AdRed
Pr
pET-3c
pUC/P450scc
P450scc
PET-3b
Bglll
Ap
EcoRI
Ap
Ap
EcoRI
Ap
EcoRI/Bglll
BamHI/EcoRI
EcoRI
Bg !!!
EcoRI
EcoRI/Rsal
BamHI
Rsal
EcoRI
Linearized pET-3b
EcoRI
BamHI
Tr
BamHI
Klenow reaction
Bglll
Pr
ligation
pET-3c
Ap
Klenow
Tr
Ilgation
pET/P450scc
P450scc
EcoRI
Ap
Pr
EcoRi /Klenow
ligation of termination sequence
Bgill
AdRed
Ap
Pr
(Ba/Bgll )
Tr
pET/AdRed
AdRed
Ap
Pr
(Ba/Bgll )
A
MARKERS
MARKERS
C
5
10
25
5
10
25
+
+
4
AdRed
+
+
+
69
116
97.4
66
46-
45
30-
B
P450scc
MARKERS
5
10
25
D
97.4
+
+
+
66
97.4
45
69
31
46
21.5
30
tion without further purification. Purified human Adx (1 mg) was a generous gift from Dr. Larry Vickery (University of California, Ir- vine, CA). The bacterial expression and purification of this protein have been previously described (28).
Antibody production
Approximately 500 µg partially purified P450scc and AdRed and purified Adx were each injected intradermally into two New Zealand White rabbits. The same amounts were then given as a booster injection at 6 weeks, and blood was collected 14 days later. Serum was seperated from the clotted blood, stored at -80 C, and used without further purification.
Cell culture
JEG-3 human choriocarcinoma cells and Y-1 mouse adrenocortical tumor cells were obtained from the American Type Culture Collection (Rockville, MD). Established conditions were used to grow the JEG-3 (8) and Y-1 (23) cells. Hormonal incubations with 1 mM 8Br-cAMP, 30 nM phorbol 12-myristate 13-acetate (PMA, also known as TPA; Sigma, St. Louis, MO), and 1 AM A23187 (Sigma) were performed as previously described (23).
Mitochondrial preparation and protein analysis
Cells were washed in PBS, scraped from the plate with a rubber policeman, and pelleted at 1,000 × g for 2 min. The cells were resus- pended in 1 ml mitochondrial sucrose buffer [0.25 M sucrose, 50 mM ethanolamine, 10 mm Tris-HCI (pH 7.4), and 1 mm EDTA] and sonicated with two 5-sec bursts using a sonicator (Artek Systems Corp.) at a setting of 20. Cell debris was removed by centrifugation for 10 min at 1,000 x 8. The supernatant was then centrifuged at 12,000 x g for 10 min; the mitochondrial pellet was washed with 1.0 ml ice-cold 0.1 M potassium phosphate (pH 7.4), recentrifuged, and suspended in 200 ul phosphate buffer. Samples containing 25 µg mitochondrial protein were electro- phoresed in SDS-12% polyacrylamide gels for P450scc or AdRed and in 18% polyacrylamide gels for Adx. Gels were transferred onto nitrocel- lulose membranes (Schleicher and Shuell, Keene, NH) overnight in 20 mM Na2HPO4-20% MeOH, and Western blotting was carried out, as previously described (29), using the antisera (1:1,000 dilution for P450scc, 1:250 for Adx, and 1:200 for AdRed). The protein bands were seen by labeling the filters with [125]]protein-A.
RIA
Cholesterol side-chain cleavage activity was measured by pregneno- lone formation using a RIA, essentially as previously described (30). The culture medium (1 or 2 ml) was extracted with 10 vol diethyl ether, and the extract was dried under nitrogen, then purified by partition chro- matography on System II Celite microcolumns by stepwise elution with isooctane (3.5 ml) and 5% ethyl acetate in isooctane (2 ml). Microcolumns were prepared by packing 2 g diatomaceous earth (Sigma) into 5-ml pipettes. The samples were dried under nitrogen, resuspended in assay buffer, and incubated with antipregnenolone antiserum and [3H]preg- nenolone (both from ICN Biomedicals, Inc., Carson, CA) for 16 h at 4 C. Unbound pregnenolone was adsorbed with charcoal and centrifuged at 3000 x g for 15 min at 4 C, and the supernatant was counted in a liquid scintillation counter. All samples were assayed in triplicate. Inter- and intraassay variations were less than 10%. Data are reported as the mean ± SEM of three experiments assayed in triplicate, and statistical comparisons were performed with paired t tests.
Results
Production of antibodies
We have used a variety of systems to produce steroido- genic enzymes in bacteria, yeast, and mammalian cells and have consistently achieved expression of the largest amount of protein with bacterial pET vectors (26). These vectors contain the promoter from gene 10 of bacteriophage T7; this promoter can be transcribed at very high rates, which, in the case of P450scc, yields 75% of the total cellular protein. The human cDNAs for P450scc and AdRed were truncated by cleavage at convenient restriction sites to remove sequences encoding the leader peptides and first few amino-terminal amino acids (Fig. 1). These truncated cDNAs were inserted in the correct reading frame in either pET-3b (P450scc) or pET-3c (AdRed). These vectors were then transformed into E. coli BL21(DE3)plysS, a strain that overproduces T7 RNA polymerase encoded by the corresponding T7 gene cloned under control of the lac UV5 promoter (31). When the
A
MARKERS
HOURS 8Br - CAMP
MARKERS
HOURS PMA/A23187
0 2 4 8 12 24
0 2 4 8 12 24
97.4
69.
46.
30.
B
MARKERS
HOURS 8Br - CAMP
MARKERS
HOURS PMA/A23187
0 2 4 8 12 24
0
2 4 8 12 24
46.
30.
21.5
14.3 -
C
MARKERS
HOURS 8Br - CAMP
MARKERS
HOURS PMA/A23187
0 2 4 8 12 24
0 2 4 8 12 24
97.4 -
69.
46.
30.
bacteria are induced with IPTG, which stimulates the lac promoter, T7 polymerase accumulates and stimulates tran- scription from the bacteriophage T7 gene 10 promoter in the pET vector, thus producing large quantities of mRNA encod- ing P450scc or AdRed. As a result, the bacteria accumulate P450scc or AdRed sequestered in inclusion bodies, which
7
CAMP
6
pregnenolone(ng/ml)
5
4
control
3
2
PMA/A23187
1
£
0
0
4
8
12
16
20
2 4
time (h)
can be readily purified. The corresponding protein can be purified nearly to homogeneity by electrophoresis through a single SDS-polyacrylamide gel (Fig. 2, A and B). Western blotting confirms that bacteria harboring pET/AdRed (Fig. 2C) or pET/P450scc (Fig. 2D) made small amounts of AdRed or P450scc without induction by IPTG, but made large amounts of these proteins when induced with 0.4 mM IPTG. The 52-kilodalton P450scc band (Fig. 2D) and the 48-kilo- dalton AdRed band (Fig. 2C) are slightly smaller than the authentic human proteins, as the pET constructions made P450scc lacking 23 N-terminal amino acids and AdRed lack- ing 8 N-terminal amino acids. These deletions did not alter either the antigenicity of the proteins or the specificity of the antibodies raised against them.
The antibodies to human P450scc, Adx, and AdRed were highly specific. They detected single bands of protein on Western blots probed with each antibody (Fig. 3). The anti- bodies to Adx and AdRed detected small amounts of these proteins in all tissues and cell lines examined, consistent with their established roles as generic electron transfer proteins for all mitochondrial forms of cytochrome P450 in all tissues. By contrast, P450scc protein was detected only in steroido- genic cells, but not in COS-1 or Hela cells (not shown).
Characterization of the cholesterol side-chain cleavage system in JEG-3 cells
We used the antibodies to human P450scc, Adx, and AdRed to examine the abundance of the corresponding proteins in human JEG-3 choriocarcinoma cells and mouse Y-1 adrenocortical tumor cells stimulated with activators of both the PKA and PKC pathways. Cells were incubated with 1 mM 8Br-cAMP or with a combination of 30 nm PMA plus 1 AM A23187 for 0, 2, 4, 8, 12, or 24 h, and the cellular contents of P450scc, Adx, and AdRed were examined by Western blotting. We then compared the abundance of these proteins to cholesterol side-chain cleavage activity, as indi- cated by the conversion of cholesterol to pregnenolone. Cholesterol was from lipoproteins in the serum added to the
culture medium and from endogenous cellular stores.
In human JEG-3 choriocarcinoma cells, P450scc accumu- lated dramatically (Fig. 3A), and Adx accumulated slightly (Fig. 3B) in response to 8Br-cAMP and diminished in re- sponse to PMA/A23187. By contrast, AdRed was unaffected by either treatment protocol over the course of 24 h (Fig. 3C). The concentration of pregnenolone in the culture me- dium was about 3-fold higher after 24 h of stimulation with 8Br-cAMP compared to the control value (0.01 < P < 0.02), consistent with the cAMP-induced increases in P450scc and Adx, while PMA/A23187 suppressed pregnenolone secretion to about one third of the control value after 24 h of incubation (0.01<P <0.02; Fig. 4).
Characterization of the cholesterol side-chain cleavage system in Y-1 cells
In mouse Y-1 adrenocortical carcinoma cells, 8Br-cAMP stimulated increases in P450scc (Fig. 5A), Adx (Fig. 5B), and AdRed (Fig. 5C). By contrast, TPA/A23187 diminished P450scc substantially and Adx slightly, but had no effect on AdRed over the 24-h time course of the experiment. The concentration of pregnenolone in the culture medium was about 9-fold higher after 24 h of stimulation with 8Br-cAMP (0.005 < P < 0.0025), consistent with the tropic effect on all three components of the side-chain cleavage system, while incubation with PMA did not reduce the low level of basal Y-1 cell pregnenolone secretion compared to that in unstim- ulated control cells (P < 0.5; Fig. 6).
Substrate for cholesterol side-chain cleavage
Conversion of cholesterol to pregnenolone is the rate- limiting step in steroidogenesis (32), but it is less clear if the limiting step is access of substrate to the enzyme or enzymatic function per se. To examine this question in JEG-3 cells, we compared the production of pregnenolone by cells receiving their cholesterol substrate from serum lipoproteins and en- dogenous stores to that by cells incubated with 22-hydrox- ycholesterol for 24 h, which is soluble and freely diffusable. JEG-3 cells incubated with 5 uM 22-hydroxycholesterol for 24 h produced 210 ± 31 ng/ml pregnenolone, while control cells produced 25.4 ± 3.8 ng/ml pregnenolone (the numbers differ from those in Fig. 4 because of differences in cell number and density in the cultures). These data suggest that access to substrate can be a limiting step in steroidogenesis.
Discussion
Steroidogenesis is acutely and chronically regulated by ACTH and gonadotropins via the PKA pathway and by angiotensin-II via the PKC pathway. The effects of these hormones and second messengers on steroidogenesis and on the mRNAs and genes for steroidogenic enzymes have been characterized in many systems, but less is known about the effects of these agents on the proteins themselves. The abun- dance of P450scc protein and the amount of pregnenolone produced in both the Y-1 and JEG-3 cell lines correlated closely. This parallelism between P450scc protein and ste-
A
MARKERS
MARKERS
HOURS 8Br - CAMP
HOURS PMA/A23187
0
2 4 8 12 24
0 2 4 8 12 24
97.4 -
69.
46.
30.
B
MARKERS
MARKERS
HOURS 8Br - CAMP
HOURS PMA/A23187
0 2 4 8 12 24
0 2 4 8 12 24
30.0 -
21.5-
14.3-
C
MARKERS
HOURS 8Br - CAMP
MARKERS
HOURS PMA/A23187
0 2 4 8 12 24
0 2 4 8 12 24
97.4 -
69.
46.
30.
roidogenesis is similar to that between P450scc mRNA and progesterone synthesis in luteinized human granulosa cells chronically stimulated with hCG (11) or in primary cultures of human fetal adrenal cells stimulated with ACTH or cAMP (18, 33). In general, activators of the PKA pathway chroni- cally stimulate steroidogenesis by stimulating transcription
20
CAMP
16
Pregnenolone(ng/ml)
12
8
PMA/A23187
control
4
0
0
4
8
1 2
16
20
2 4
time (h)
of the genes encoding the various enzymes. However, the action of cAMP can extend beyond gene transcription. We recently demonstrated that cAMP posttranscriptionally de- creases the abundance of AdRed mRNA in JEG-3 cells (10), although in both that study and in Fig. 3c, cAMP had no detectable effect on AdRed protein after 24 h, suggesting that this enzyme is stable and has a long half-life. P450scc gene transcription, mRNA abundance, protein abundance, and side-chain cleavage activity all appear to be stimulated in parallel by cAMP in both JEG-3 and Y-1 cells (Refs. 23 and 25 and present study). By contrast, the regulation of Adx appears to be complex and varied in different cells. We found no induction of Adx gene transcription measured by RNA polymerase run-on assays in JEG-3 cells (34), although the mRNA accumulates in response to cAMP (8, 19, 34), sug- gesting a posttranscriptional mechanism. Our present studies show that Adx protein accumulates in response to cAMP in both JEG-3 and Y-1 cells, consistent with the observations on its mRNA abundance.
The responses of the steroidogenic machinery to activators of the PKC pathway have been studied in less detail. Angio- tensin-II, which stimulates aldosterone secretion through the PKC second messenger pathway, elicits a biphasic steroido- genic response. It acutely stimulates aldosterone secretion, but chronic exposure to angiotensin-II diminishes aldoste- rone secretion (16). Similarly, promoter/reporter construc- tions of the human P450scc gene exhibit transient transcrip- tional stimulation by PMA and A23187 in Y-1 cells (23). However, exposure for 24 h reduces transcription of these promoter/reporter constructions to about 20% of the control value and reduced endogenous Y-1 cell P450scc mRNA to a similar level (23). We found a decrease in P450scc protein in response to PMA and A23187 in our present studies, al- though the secretion of pregnenolone remained similar to that in controls. Similarly, although P450c17 (17a-hydrox- ylase/17,20-lyase) is not involved in the steroidogenic re- sponse to angiotensin-II, it is also suppressed by the PKC pathway. When human P450c17 promoter/reporter con-
structions are transfected into Y-1 cells, their transcription is suppressed by PMA (24), and accumulation of P450c17 mRNA in primary cultures of human fetal adrenal cells is similarly suppressed (35). Thus, the actions of the PKC pathway on steroidogenesis are likely to be rather complex and require considerable further investigation.
Finally, our data suggest that the availability of substrate is a rate-limiting step in steroidogenesis. 22-R-Hydroxycho- lesterol, which bypasses the normal cholesterol transport apparatus results in a 10-fold higher level of pregnenolone over 24 h. This suggests that only a small subset of the total cellular cholesterol is available for conversion to pregneno- lone and that the cholesterol in the inner mitochondrial membrane probably does not supply the cholesterol used for steroid hormone biosynthesis (36). Thus, other mechanisms in addition to regulation of the components of the cholesterol side-chain cleavage system play an important role in regu- lating the activity of this system and, thus, regulating steroi- dogenesis.
Acknowledgments
We thank Bruce Heyer in the Division of Pediatric Endocrinology for consultation in setting up the pregnenolone RIA, and Dr. Julien I. E. Hoffman for consultation concerning the statistical analysis of the RIA data.
References
1. Miller WL 1988 Molecular biology of steroid hormone synthesis. Endocr Rev 9:295-318
2. Hanukoglu I, Suh BS, Himmelhoch S, Amsterdam A 1990 Induc- tion and mitochondrial localization of cytochrome P450scc system enzymes in normal and transformed ovarian granulosa cells. J Cell Biol 111:1373-1381
3. Morohashi K, Sogawa K Omura T, Fujii-Kuriyama Y 1987 Gene structure of human cytochrome P-450scc, cholesterol desmolase. J Biochem 101:879-887
4. Chang C-Y, Wu D-A, Lai C-C, Miller WL, Chung B 1988 Cloning and structure of the human adrenodoxin gene. DNA 7:609-615
5. Lin D, Shi Y, Miller WL 1990 Cloning and sequence of the human adrenodoxin reductase gene. Proc Natl Acad Sci USA 87:8516-8520
6. Sparkes RS, Klisak I, Miller WL 1991 Regional mapping of genes encoding human steroidogenic enzymes: P450scc to 15q23-q24, adrenodoxin to 11q22; adrenodoxin reductase to 17q24-q25; and P450c17 to 10q24-q25. DNA Cell Biol 10:359-365
7. Chung B, Matteson KJ, Voutilainen R, Mohandas TK, Miller WL 1986 Human cholesterol side-chain cleavage enzyme, P450scc: cDNA cloning, assignment of the gene to chromosome 15, and expression in the placenta. Proc Natl Acad Sci USA 83:8962-8966
8. Picado-Leonard J, Voutilainen R, Kao L, Chung B, Strauss JF III, Miller WL 1988 Human adrenodoxin: cloning of three cDNAs and cycloheximide enhancement in JEG-3 cells. J Biol Chem 263:3240- 3244 (corrected 11016)
9. Solish SB, Picado-Leonard J, Morel Y, Kuhn RW, Mohandas TK, Hanukoglu I, Miller WL 1988 Human adrenodoxin reductase: two mRNAs encoded by a single gene on chromosome 17cen-+q25 are expressed in steroidogenic tissues. Proc Natl Acad Sci USA 85:7104- 7108
10. Brentano ST, Black SM, Lin D, Miller WL 1992 cAMP post- transcriptionally diminishes the abundance of adrenodoxin reduc- tase mRNA. Proc Natl Acad Sci USA 89:4099-4103
11. Voutilainen R, Tapanainen J, Chung B, Matteson KJ, Miller WL 1986 Hormonal regulation of P450scc (20,22-desmolase) and P450c17 (17a-hydroxylase/17,20 lyase) in cultured human granu- losa cells. J Clin Endocrinol Metab 63:202-207
12. Voutilainen R, Miller WL 1986 Developmental expression of genes for the steroidogenic enzymes P450scc (20,22 desmolase), P450c17 (17a-hydroxylase/17,20 lyase), and P450c21 (21-hydroxylase) in the human fetus. J Clin Endocrinol Metab 63:1145-1150
13. Voutilainen R, Miller WL 1988 Developmental and hormonal regulation of mRNAs for insulin-like growth factor and steroido- genic enzymes in human fetal adrenals and gonads. DNA 7:9-15
14. Miller WL 1989 Regulation of mRNAs for human steroidogenic enzymes. Endocr Res 15:1-16
15. Waterman MR, Simpson ER 1989 Regulation of steroid hydroxyl- ase gene expression is multifactorial in nature. Recent Prog Horm Res 45:533-566
16. Barrett PQ, Bollag WB, Isales CM, McCarthy RT, Rasmussen H 1989 The role of calcium in angiotensin II-mediated aldosterone secretion. Endocr Rev 10:496-518
17. Voutilainen R, Miller WL 1987 Coordinate tropic hormone regu- lation of mRNAs for insulin-like growth factor II and the cholesterol side-chain cleavage enzyme, P450scc, in human steroidogenic tis- sues. Proc Natl Acad Sci USA 84:1590-1594
18. DiBlasio AM, Voutilainen R, Jaffe RB, Miller WL 1987 Hormonal regulation of mRNAs for P450scc (cholesterol side-chain cleavage enzyme) and P450c17 (17a-hydroxylase/17,20 lyase) in cultured human fetal adreanal cells. J Clin Endocrinol Metab 65:170-175
19. Voutilainen R, Picado-Leonard J, DiBlasio AM, Miller WL 1988 Hormonal and developmental regulation of human adrenodoxin mRNA in steroidogenic tissues J Clin Endocrinol Metab 66:383-388
20. Tremblay Y, Ringler GE, Morel Y, Mohandas TK, Labrie F, Strauss III JF, Miller WL 1989 Regulation of the gene for estrogenic 17-ketosteroid reductase lying on chromosome 17cen→q25. J Biol Chem 264:20458-20462
21. Moore CCD, Miller WL 1991 The role of transcriptional regulation in steroid hormone biosynthesis. J Steroid Biochem Mol Biol 40:517- 525
22. Hum DW, Miller WL, Transcriptional regulation of human genes for steroidogenic enzymes. Clin Chem, in press
23. Moore CCD, Brentano ST, Miller WL 1990 Human P450scc gene transcription is induced by cyclic AMP and repressed by 12-O- tetradecanolyphorbol-13-acetate and A23187 by independent cis- elements. Mol Cell Biol 10:6013-6023
24. Brentano ST, Picado-Leonard J, Mellon SH, Moore CCD, Miller WL 1990 Tissue-specific cAMP-induced and phorbol ester repressed expression from the human P450c17 promoter in mouse cells. Mol Endocrinol 4:1972-1979
25. Moore CCD, Hum DW, Miller WL 1992 Identification of positive and negative placenta-specific basal elements and a cyclic adenosine 3’,5’-monophosphate response element in the human gene for P450scc. Mol Endocrinol 6:2045-2058
26. Studier FW, Rosenberg AH, Dunn JJ, Dubendorf JW 1990 Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60-89
27. Hager DA, Burges RR 1980 Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topisomerase, and other enzymes. Anal Biochem 109:76-86
28. Coghlan VM, Vickery LE 1989 Expression of human ferredoxin and assembly of the [2Fe-2S] center in Escherichia coli. Proc Natl Acad Sci USA 86:835-839
29. Adams DJ, Seilman S, Amelizad Z, Oesch F, Wolf CR 1985 Identification of human cytochromes P450 analogous to forms induced by phenobarbital and 3-methylcholanthrene in the rat. Biochem J 232:869-876
30. Abraham GE, Manlimos FS, Garza A 1977 Radioimmunoassay of steroids. In: Abraham GE (ed) Handbook of Radioimmunoassay. Marcel Dekker, New York, pp 591-656
31. Studier FW, Moffatt BA 1986 Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113-130
32. Stone D, Hechter O 1955 Studies on ACTH action in perfused bovine adrenals: aspects of progesterone as an intermediary in cortico-steroidogenesis. Arch Biochem Biophys 54:121-128
33. Mellon SH, Shively JE, Miller WL 1991 Human proopiomelano- cortin (79-96), a proposed androgen stimulatory hormone, does not affect steroidogenesis in cultured human fetal adrenal cells. J Clin Endocrinol Metab 72:19-22
34. Brentano ST, Miller WL 1992 Regulation of human P450scc and adrenodoxin messenger ribonucleic acids in JEG-3 cytotrophoblast cells. Endocrinology 131:3010-3018
35. Voutilainen R, Ilvesmaki V, Miettinen PJ 1991 Low expression of 38-hydroxy-5-ene steroid dehydrogenase gene in human fetal ad- renals in vivo; adrenocorticotropin and protein kinase C-dependent regulation in adrenocortical cultures. J Clin Endocrinol Metab 72:761-767
36. Stevens VL, Xu T, Lambeth JD 1992 Cholesterol pools in rat adrenal mitochondria: use of cholesterol oxidase to infer a complex pool structure. Endocrinology 130:1557-1563