REGULATION OF THE ADRENAL STEROIDOGENIC ENZYMES 
Keith L. Parker, M.D., Ph.D., Assistant Investigator 
Dr. Parker's laboratory is studying the mecha- 
nisms that control the gene expression of the adre- 
nal steroidogenic enzymes. Steroid biosynthesis, 
which involves the conversion of cholesterol to bio- 
logically active products, requires the concerted ac- 
tion of five cytochrome P-450 enzymes. Certain of 
these en2ymes, such as the side-chain cleavage en- 
2yme (SCC), are expressed in all steroidogenic tis- 
sues; others, notably steroid 21-hydroxylase (21- 
OHase) and lip-hydroxylase (lip-OHase), are 
expressed only in the adrenal cortex. Treatment of 
adrenocortical cells with the trophic hormone 
ACTH coordinately increases the expression of all 
of these genes via the cAMP second messenger sys- 
tem. This induction represents a major component 
of the hormone's action to maintain adrenocortical 
competence for steroid biosynthesis. The objective 
of these studies is to define the factors that regulate 
the expression of the adrenal steroidogenic en- 
zymes. A central hypothesis is that shared factors 
determine the ACTH induction and adrenal-specific 
expression of this network of genes, whereas 
unique elements permit SCC expression in other 
steroidogenic tissues. 
I. 21 -Hydroxylase Gene Regulation. 
Initial studies focused on 21-OHase. Transfection 
experiments showed that all elements essential for 
tissue-specific and cAMP-inducible expression were 
found within 330 bp of the transcription start site. 
Functional and DNase I footprinting experiments 
defined a complex array of regulatory elements 
within these 330 bp. Two of these elements, at -65 
and -215, share the heptamer AGGTCAG and bind 
the same, or highly related, protein(s). Moreover, 
both elements increased the activity of a heterolo- 
gous promoter in an adrenal-specific manner. 
Ongoing studies are directed at characterizing 
the proteins that interact with these elements to 
regulate 21-OHase transcription. These studies, 
which will focus on the protein that interacts with 
the AGGTCAG motif, will utilize two complemen- 
tary approaches: direct purification of the regula- 
tory proteins and screening of expression libraries 
with oligonucleotide probes. To provide sufficient 
material to make the first approach possible, meth- 
ods have been developed for making whole-cell ex- 
tracts from bovine adrenal glands. These prepara- 
tions appear to contain most, if not all, of the 
proteins previously characterized in Yl cells. The 
first step in the expression screening of cDNA li- 
braries with oligonucleotide probes is the construc- 
tion of the libraries in Xgtll. Such libraries have 
been prepared from both mouse adrenal RNA and 
Yl cell RNA, and screening with oligonucleotides is 
now in progress. 
To extend their investigations of 21-OHase ex- 
pression in Yl cells, Dr. Parker and his colleagues 
recently initiated a collaboration with Drs. Jona- 
than G. Seidman (HHMI, Harvard Medical School) 
and David Milstone to study expression of 21- 
OHase in transgenic mice. These experiments sug- 
gest that additional regulatory elements that are 
not required for expression in Yl cells are required 
for 21-OHase expression in transgenic mice. After 
the region that controls this effect is mapped, stud- 
ies will be performed to determine if any adrenal- 
specific differences, such as DNase I-hypersensitive 
sites or differences in methylation status, are 
unique to that region. 
II. lip-Hydroxylase Gene Regulation. 
To complement and extend the studies with 21- 
OHase, the laboratory cloned and characterized the 
llp-OHase gene. As was the case for 21-OHase, the 
5 '-flanking region of ll(3-OHase directed adrenal- 
specific and cAMP-induced expression of a linked 
reporter gene. Potential regulatory elements within 
the lip-OHase promoter region were defined by 
DNase I footprinting experiments with nuclear ex- 
tracts from Yl adrenocortical cells. One interaction, 
centered at -52, involved a sequence that closely 
resembled the consensus sequence of the cAMP-re- 
sponsive element, an element found in the pro- 
moter region of many cAMP-responsive genes. Ex- 
tensive studies showed that this element, which is 
essential for ll(3-OHase expression, determines 
cAMP responsiveness of the gene in a manner anal- 
ogous to the cAMP-responsive element defined in 
other systems. DNase I footprinting analyses de- 
fined two other elements, one centered at -310 
and one at -370. Mutation of either sequence 
significantly inhibited promoter activity. Moreover, 
oligonucleotides containing either element re- 
stored activity, in an orientation-independent man- 
ner, to a promoter fragment containing 1 1 ^OHase 
sequences from -40. Examination of the sequence 
of the 5 '-flanking region of the llfi-OHase gene 
Continued 
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