(Received for publication, June 5, 1995; and in revised form, August 21, 1995)
From the
Two sequence elements located at -111 to -100 base pairs and -70 to -50 base pairs in the 5`-flanking region of the bovine CYP11A gene and in closely related positions in CYP11A of other species contain G-rich regions that are similar to the consensus Sp1-binding site. These sequences bind the purified transcription factor Sp1 as well as nuclear proteins from mouse Y1 adrenal cells that interact with an antibody specific for Sp1. Both of these CYP11A sequences support basal and cAMP-dependent transcription of reporter gene plasmids transfected into Y1 cells, and mutations within the G-rich -111/-100-base pair sequence that reduce or eliminate the binding of Sp1-related Y1 nuclear proteins also markedly reduce cAMP-induced transcription. cAMP-dependent transcription supported by both CYP11A sequence elements is mediated by protein kinase A at levels comparable to that promoted by different cAMP-response sequences and transcription factors in other genes involved in steroidogenesis. These results indicate that ACTH-dependent regulation of cholesterol side chain cleavage cytochrome P450 levels in the adrenal cortex which is mediated through cAMP involves the ubiquitous transcription factor Sp1.
In the adrenal cortex of vertebrate species, synthesis of glucocorticoids, mineralocorticoids, and precursors of sex hormones is catalyzed by steroid hydroxylases which are members of the cytochrome P450 gene superfamily (Simpson and Waterman, 1988). The initial and rate-limiting step in steroid hormone biosynthesis is the conversion of cholesterol to pregnenolone by the mitochondrial steroid hydroxylase cholesterol side chain cleavage cytochrome P450 which is the product of CYP11A gene (Nelson et al., 1993). The transcription of this gene along with three other steroid hydroxylase genes, namely CYP11B1, CYP17, and CYP21, is coordinately regulated in the bovine adrenal cortex by the peptide hormone corticotropin (ACTH) via cAMP (John et al., 1986). Constitutive expression of CYP11A, in the absence of ACTH, is also observed in the adult bovine adrenocortical cells (John et al., 1984), fetal bovine adrenal cortex (Lund et al., 1988), and fetal human adrenal cells (John et al., 1987).
Functional analyses of the 5`-flanking regions of
CYP11A genes from human (Inoue et al., 1988; Moore et
al., 1990, 1992; Hum et al., 1993; Guo et al.,
1994), bovine (Ahlgren et al., 1990; Momoi et al.,
1992), rat (Oonk et al., 1990; Clemens et al., 1994),
and mouse (Rice et al., 1990) have revealed species-specific
variation in the composition of cAMP-responsive regions within this
gene as well as in their ability to support cAMP-induced transcription
in cell lines originating from different steroidogenic tissues. Serial
deletions of the proximal 896 base pairs (bp) ()of the
5`-flanking region of the bovine CYP11A gene identified a cAMP-response
sequence (CRS) between -183 and -83 bp (Ahlgren et
al., 1990). Subsequently, this element was found to reside within
-118 and -100 bp and was shown to be sufficient for
cAMP-dependent transcription of a reporter gene in mouse adrenocortical
Y1 tumor cells (Momoi et al., 1992) as well as in bovine
ovarian luteal cells (Begeot et al., 1993). This G-rich
sequence, conserved at similar positions in the CYP11A genes of
different species, has also been implicated in the negative regulation
of forskolin-induced bovine CYP11A gene transcription by phorbol esters
in bovine ovarian luteal cells (Begeot et al., 1993; Lauber et al., 1993). It does not share homology with the well
characterized cAMP-response element (CRE) (Montminy et al.,
1990) but contains a site that is similar to the consensus Sp1 binding
sequence (Kadonaga et al., 1986). The human CYP11A gene does
contain a sequence similar to the CRE between -1654 and
-1648 (Inoue et al., 1988; Moore et al., 1992;
Guo et al., 1994) which is far upstream from the G-rich region
sharing homology with bovine CYP11A.
Herein we demonstrate that 1) one of the proteins binding to the sequence between -111 and -100 bp within the bovine CYP11A promoter is Sp1 or a protein antigenically related to it, 2) mutations in this region either eliminate or markedly reduce both binding of Sp1 and cAMP-dependent transcription mediated by this element in Y1 adrenocortical cells, 3) there is at least one additional sequence between -70 and -50 bp of the bovine CYP11A gene which also binds Sp1 and supports cAMP-induced transcription, and 4) the cAMP-induced transcription mediated by the Sp1-binding sequences of the bovine CYP11A gene is dependent on the cAMP-dependent protein kinase (PKA) catalytic subunit.
The
double-stranded bovine CYP11A wild type and mutant oligonucleotide
sequences with SacI and SalI ends were inserted into
the unique SacI and SalI sites of the OVEC vector to
generate various bovine CYP11A promoter-OVEC plasmids (Fig. 1C). The OVEC construct having multiple tandem
copies of -118/-100 bp from bovine CYP11A was made
according to the scheme described by Westin et al.(1987). The
OVEC plasmids with one and two tandem copies of the human
-126/-113 CYP21 sequence, one and two tandem copies of the
bovine -243/-225 CYP17 sequence, and one copy of the bovine
699/740 adrenodoxin gene sequence are designated as 1 and 2
-126/-113h21OV (Kagawa and Waterman, 1992), 1
and 2
-243/-225b17OV (Lund et al.,
1990), and 699/740ADXOV (Chen and Waterman, 1992), respectively. The
plasmid 4
CREOV (Lund et al., 1990) contains four
tandem copies of the CRE from the human chorionic gonadotropin-
gene (Deutsch et al., 1987). Plasmids OVECREF and SV40OVEC,
containing the 72-bp repeat SV40 enhancer elements, served as an
internal reference control and a positive
-globin reporter gene
expression control, respectively.
Figure 1:
Schematic diagrams of
the bovine CYP11A proximal promoter region and its sequences used in
the reporter gene plasmids. A, the 5`-flanking bovine CYP11A
gene sequence from -118 to -32. The G-rich sequences are
boxed in rectangles while a known SF-1 binding site is encircled by an oval. The solid line above and the dashed line below mark sequences that show strong homology to the consensus
binding sites for ASP and AP-1, respectively. B, wild type and
mutant -118/-100 promoter sequences. The arrows indicate base changes in the wild type sequence. The wild-type
G-rich sequence is shown inside the rectangle. C,
OVEC--globin reporter constructs. The CYP11A promoter sequences
shown in B were inserted between SacI (ScI)
and SalI (SlI) sites in front of a minimal
-globin promoter. D, CYP11A-luciferase reporer plasmids.
CYP11A promoter fragments that contained homologous TATA sequences were
inserted into XhoI (XhI) and PstI (PsI) sites of the A
LLUC vector 5` to the
luciferase coding region. Three tandemly repeated boxes represent SV40 polyadenylation sites preceding the promoter
insertion site.
Luciferase reporter gene
constructs (Fig. 1D) -186/+12LUC, and
-101/+12LUC were made by inserting XhoI/PstI fragments from -186CATSCC, and
-101CATSCC (Ahlgren et al., 1990) into XhoI/PstI sites of the pALLUC vector. The
-186/+12LUC and -101/+12LUC contained the
homologous bovine CYP11A TATA element. All plasmid constructions were
confirmed by restriction digestion and dideoxy sequencing. The
metallothionein promoter-controlled expression vectors for catalytic
and mutant type I regulatory subunits of PKA, CEV
Neo, and
Mt-REV(AB)-Neo, respectively, were kindly provided by Dr. Stanley
McKnight, University of Washington, Seattle.
Figure 2:
Gel mobility shift analysis of binding of
Y1 nuclear proteins and purified Sp1 to wild type and mutant
-118/-100 sequences. P-Labeled double-stranded
oligonucleotides were incubated with 25 µg of Y1 nuclear proteins
or 7.5 ng of purified Sp1. In the competition assays, 10 pmol (100-fold
molar excess) of unlabeled oligonucleotides were added along with the
probe. DNA-protein complexes were analyzed by electrophoresis on a
native 4% polyacrylamide gel. A, DNA-protein complexes formed
by the labeled -118/-100 CYP11A and consensus Sp1
oligonucleotide sequences. Excess unlabeled consenus Sp1 (Sp1
OLIGO) and CYP11A mutant oligonucleotides -108/-107M,
-105/-104M, -116/-114M, -103/-101M
were used as competitors. P, probe; E, extract. B, labeled wild type -118/-100 and mutant
-103/-101M, -105/-104M, -108/-107M,
-116/-114M fragments were used as probes in the presence of
Y1 nuclear extracts and purified Sp1. The consensus Sp1 oligonucleotide (Sp1 OLIGO) was used as competitor. P, probe; EXT, extract. C, 2 µg of polyclonal anti-Sp1
(
-Sp1) was added to the -118/-100 CYP11A and
consensus Sp1 sequence probes in incubation mixtures containing either
Y1 nuclear extracts or purified Sp1. P, probe; EXT,
extract; SS, supershift. D, 2 µg of polyclonal
anti-Sp1 was added to Y1 nuclear extracts in the presence of
-118/-100, -111/-100, and -118/-104
CYP11A probes.
When both wild type and mutant -118/-100 fragments were used as probes only the -116/-114M showed a binding pattern similar to that observed with -118/-100 in the presence of either Y1 nuclear extracts or purified Sp1 (Fig. 2B). Formation of complexes I and II for which the consensus Sp1 oligonucleotide competed (Fig. 2A) were either markedly reduced, as observed with -103/-101M, or completely absent as seen in the case of -105/-104M and -108/-107M. All mutant fragments, however, still formed complex III with both Y1 nuclear extracts and purified Sp1. Also, the complexes formed by both the -118/-100 sequence and the consensus Sp1 oligonucleotide using the Y1 nuclear extracts interacted with an antibody raised against Sp1 transcription factor resulting in a supershift complex (Fig. 2C). A similar supershift complex is also formed when the consensus Sp1 sequence was incubated with purified Sp1 in the presence of Sp1 antibody.
The same complexes, including the supershift complex formed in the presence of Sp1 antibody, were observed when the -111/-100 sequence, which lacked seven nucleotides present at the 5`-end of the -118/100 sequence, was used as the probe (Fig. 2D). In contrast, these complexes were not observed when four nucleotides were removed from the 3`-end of the -118/-100 fragment (-118/-104). Together, the observations in Fig. 2suggest that the nucleotides present between positions -108 and -100 of the CYP11A promoter are important for the formation of DNA-protein complexes using Y1 nuclear extracts that contain Sp1 or a protein that is antigenically related to it.
Figure 3:
Analysis of wild type and mutant bovine
CYP11A promoter activity by transient reporter gene expression. A, mouse Y1 adrenal tumor cells were cotransfected with CYP11A
promoter constructs and an internal reference plasmid using the
CaPO method. After transfection, cytoplasmic RNA from cells
grown in the presence or absence of 25 µM forskolin was
analyzed by S1 nuclease digestion for the expression of
-globin
reporter messenger RNA. The results were quantified using a
PhosphorImager. The values obtained for correctly initiated transcripts
were normalized to the corresponding internal signal. The results
obtained in different experiments for each construct (OVEC,
-118/-100OV, n = 4;
-103/-101MOV, -105/-104MOV,
-108/-107MOV, and -116/-114MOV, n = 3) except in the case of -896/-32OV (n = 2) are shown as mean + S.D., error bars. B,
transcriptional activity directed by -118/-100 and
-111/-100 CYP11A sequences compared as described in A. The average values from two independent experiments are
shown.
Figure 4:
Y1
nuclear proteins associated with Sp1 bind to the CYP11A promoter region
between nucleotides -101 and -32. Y1 nuclear extracts were
incubated with labeled probes in A, -111/-100,
-101/-50, -70/-32; and B,
-118/-100, and -70/-50 as described in the
legend for Fig. 2. P, probe; E, Y1 nuclear
extract; SP1 OLIGO, consensus Sp1-binding oligonucleotide, SP1 AB or -Sp1, anti-Sp1 antibody;
-CREB, anti-CREB antibody; SS, supershift
complex. C, comparative analysis of the bovine CYP11A promoter
activity observed with -118/-100 and -70/-50
sequences: error bars indicate S.D. of the mean for three
separate experiments, performed as described in Fig. 3A.
D, luciferase activity directed by constructs containing
homologous CYP11A TATA sequence in Y1 cells. The average values from
two separate experiments is presented.
Figure 5:
cAMP-dependent transcription supported by
-118/-100 and -70/-50 sequences is mediated
through the PKA signal transduction pathway. Mutant KIN8 (A)
and normal Y1 (B) cells were cotransfected with 15 µg of
test plasmid, 1 µg of an internal reference plasmid, and 4 µg
of either PKA catalytic or mutant regulatory expression vector. After
transfection, cells were grown in the presence of 100 µM ZnSO throughout the experiment to activate the
metallothionein promoter-driven expression of PKA subunits. The
cytoplasmic RNA, isolated from both untreated(-) and
forskolin-treated cells (+), was analyzed for
-globin mRNA by
S1 nuclease digestion as described under methods and Fig. 3A. Because of the low level of transcriptional
activity in KIN8 cells, the average values from two experiments are
presented relative to the expression observed from the negative
controls OVEC (forskolin(-), and PKA CAT(-); not shown). In
the case of Y1 cells, which support a high level of cAMP-dependent
transcription, the data is presented as a percentage of activity
observed from 2
CREOV in the presence of forskolin. P,
probe; C.I., correctly initiated transcripts; REF,
internal control. PKA CAT and PKA REG represent PKA
catalytic and mutant regulatory subunit expression
vectors.
These
findings were further corroborated by data obtained upon cotransfection
of the PKA subunit into Y1 cells which, unlike KIN8, express a
functional endogenous PKA enzyme. In these cells, constructs with
either -118/-100 or -70/-50 sequences from the
bovine CYP11A promoter respond to the exogenously supplied free
catalytic subunit by supporting a 4-5-fold increased
transcriptional activity compared to that observed in the control cells (Fig. 5B). The same effect could also be reproduced by
treating the cells with forskolin alone. Conversely, when a mutant form
of the PKA regulatory subunit was expressed in these cells, forskolin
treatment did not support the same level of increased expression as
observed when the cells were treated with forskolin alone. This
suggests that exogenous mutant PKA regulatory subunit binds a portion
of the catalytic subunit that is dissociated from the endogenous wild
type regulatory PKA subunit by cAMP action. However, since the mutant
regulatory subunit lacks the ability to interact with cAMP, the bound
catalytic subunit can no longer participate in the signal transduction
pathway responsible for the activation of cAMP-dependent transcription
(Rae et al., 1979). The plasmid -105/-104MOV,
which carries mutations in the G-rich region of the
-118/-100 sequence and cannot bind Sp1, did not support
transcriptional activity in response to either forskolin treatment or
exogenously supplied PKA catalytic subunit. In contrast, 2
CREOV, the plasmid that contains two consensus CRE elements, showed a
very high level of transcriptional activity under these conditions.
Figure 6:
Comparison of transcriptional activity
supported by CRSs of different genes involved in steroidogenesis.
Transcriptional activity directed by promoter constructs containing one
(1) or two (2
) copies of discrete CRS elements from
bovine CYP11A (-118/-100bSCCOV and
-70/-50bSCCOV), bovine CYP17
(-243/-225b17OV), human CYP21
(-126/-113h21OV), and bovine adrenodoxin (699/740ADXOV) genes were compared in Y1 cells alongside CREOV
plasmids which carry one or two copies of the CRE
sequence.
In previous studies the sequence -118/-100, containing a putative binding site for the transcription factor Sp1, has been shown to be involved in the cAMP-dependent transcription of the bovine CYP11A gene in Y1 (Momoi et al., 1992) and bovine ovarian luteal (Begeot et al., 1993) cells. We now report that a minimal 12-bp G-rich sequence present between -111 and -100 can indeed bind to Sp1 or an antigenically related protein and this fragment is sufficient to support cAMP-dependent transcription at the same level as that observed with the longer -118/-100 fragment. Evidence is also presented for the identification of an additional cAMP responsive sequence in the region between -70 and -50. While mutagenesis of the putative Sp1-binding site within -70/-50 has not been carried out, all experimental features of this element including gel shift pattern with Y1 nuclear extracts or purified Sp1, supershift by anti-Sp1, and transcriptional responsiveness to both forskolin and PKA catalytic subunit are similar to -118/-100. We infer from these similarities that both -118/-100 and -70/-50 can bind Sp1 and function independently as cAMP-responsive sequences. Thus, within the proximal 110 bp of the bovine CYP11A promoter sequence, two CRS elements participate in cAMP-induced transcription in Y1 cells. Consistent with these results, the -101/+12 and -186/+12 bovine CYP11A fragments support cAMP-induced transcription in Y1 cells at levels expected from one or both of the identified Sp1-binding elements.
Among the steroidogenic genes, evidence for the existence of multiple CRS sequences has also been documented for mouse (Rice et al., 1990) and human (Guo et al., 1994) CYP11A and bovine CYP17 (Lund et al., 1990). When the available sequences from the 5`-flanking regions of bovine (Ahlgren et al., 1990), ovine (Pestell et al., 1993), mouse (Rice et al., 1990), rat (Oonk et al., 1990), and human (Morohashi et al., 1987) CYP11A genes are compared (Fig. 7), it is noticed that -111/-100 and -70/-50 sequences are among the five upstream regions of greatest sequence homology between the genes (boxed areas). There is a 100% identity in the regions -111/-100 and -70/-50 between bovine and ovine sequences, while putative Sp1-binding sites are also present in the regions of the human, mouse, and rat genes corresponding to bovine -110/-100. It is also noteworthy that the corresponding sequences within the -118/-100 and -70/-50 regions of homology have been shown to be important for the cAMP-induced transcription of mouse CYP11A in Y1 cells (Rice et al., 1990). Similarly, the human -118/-100 sequence and the rat -73/-38 sequence have also been shown to support cAMP-dependent transcription in Y1/JEG3 (Guo et al., 1994) and rat granulosa cells (Clemens et al., 1994), respectively.
Figure 7: Alignment of the sequences in the proximal promoter regions of CYP11A from different species. The sequences showing the greatest homology in the proximal promoter regions of CYP11A genes from different species are shown in the boxes. The bases that differ from the bovine sequence are underlined and shown in bold. Dots indicate the gaps introduced to maximize the sequence homology. The numbers above the boxes indicate the nucleotide positions relative to the transcriptional start site of bovine CYP11A. The 5`-end of ovine is -109, mouse is -118, rat is -119, human is -117.
It has been suggested by Rice et al.(1990) that a sequence similar to the consensus SF-1 binding site (AGGTCA) is responsible for the cAMP-induced transcription observed with the -77/-60 region of the mouse gene in Y1 cells. However, this sequence present at similar position in an identical context in the rat gene is not required for cAMP-dependent transcription in rat granulosa cells (Clemens et al., 1994). In addition, when AGGAGC, present at nucleotide positions -106 to -101 in the bovine CYP11A sequence, was changed to AGGTCA (Fig. 1B, -103/-101M), to make it identical to the SF-1 binding sequence in the mouse gene cited above, cAMP induced transcription was abolished (Fig. 3A). Whether the highly conserved sequence -60/-50, which is nearly identical in the CYP11A genes from different species (Fig. 7), plays a more important part than the GC-rich -70/-60 sequence in conferring the cAMP responsiveness to the proximal bovine CYP11A promoter (-70/-50) remains to be studied.
There
are other examples of genes that contain Sp1-binding sites either close
to or within a cAMP-responsive element. These include genes that encode
human CYP21 (Kagawa and Waterman, 1992), type II cAMP-dependent
protein kinase regulatory subunit (Kurten et al., 1992),
bovine adrenodoxin (Chen and Waterman, 1992), human ferredoxin (Chang et al., 1992), and human urokinase (Grimaldi et al.,
1993). Although, the importance of Sp1 in the constitutive expression
of genes, in general, is well understood, the evidence for its direct
role in cAMP-mediated transcription has not yet been clearly
established at a mechanistic level. In the case of the human CYP21
gene, Sp1 and the adrenal-specific protein (ASP) have overlapping
binding sites in the -129/-96 sequence, although ASP alone
can support cAMP-dependent transcription from a shorter
-126/-113 promoter fragment (Kagawa and Waterman, 1992).
Interestingly, ASP also binds to the bovine CYP11A sequence around
-101 to -89 (Momoi et al., 1992) but is not
involved in cAMP-dependent transcription of this gene. In another
example, a sequence similar to the consensus Sp1-binding site in the
cAMP-responsive element (-54/-42) of the human urokinase
gene interacts with cAMP-induced DNA-protein complexes in mouse Sertoli
cells (Grimaldi et al., 1993). In other genes, evidence for
the involvement of Sp1 is also documented in the case of retinoic
acid/cAMP-dependent and differentiation-specific transcription of the
tissue plasminogen activator gene (Darrow et al., 1990).
Acting through retinoblastoma control elements, Sp1 has also been shown
to activate transcription of early response genes such as c-fos (Udvadia et al., 1993), the expression of which is
induced by ACTH (Miyamoto et al., 1992).
In genes such as
CYP11A and adrenodoxin which are expressed constitutively, the former
being active exclusively or predominantly in steroidogenic tissues, the
general transcription factor Sp1 might play a very important role in
maintaining the basal transcription. Indeed, each cAMP-responsive
sequence of the bovine CYP11A gene (-118/-100 and
-70/-50) which has one Sp1-binding site and the bovine
adrenodoxin sequence from 699 to 740 which contains two Sp1 sites
confer basal expression to a -globin reporter gene in Y1 cells (Fig. 6). However, it is also noticed that these same sequences
also support cAMP-dependent transcription (Fig. 6) and any
mutations that reduce Sp1 binding to the -118/-100 sequence
in bovine CYP11A gene reduce, in parallel, both basal and cAMP-induced
transcription (Fig. 2B and Fig. 3A).
Thus, these multiple dual role responsive sequences of bovine CYP11A,
just as in their murine counterparts (Rice et al., 1990),
might function independently or in association in supporting optimal
level of CYP11A transcription.
Coexpression studies of PKA catalytic and regulatory subunits in KIN8 and Y1 cells show that the bovine CYP11A cAMP-responsive sequences -118/-100 and -70/-50 function via a cAMP-PKA signal transduction pathway. Since Sp1 does not have a PKA phosphorylation site and at least in -111/-100 an Sp1-like protein is the only one which binds directly to the DNA, regulation of transcription directed by the G-rich elements at -111/-100 and -70/-50 sequences probably involves Sp1 or an Sp1-like factor in combination with an as yet unidentified cell-specific protein. This unknown protein would be predicted to interact directly with Sp1 and to not be a DNA-binding protein. Perhaps it could function like CBP, the nuclear protein which serves as a coactivator for the phosphorylated form of CREB (Chrivia et al., 1993; Kwok et al., 1994). However, in this case the unknown coactivator might indeed be a target for PKA in execution of its role in coupling the ubiquitous transcription factor Sp1 with cAMP-dependent activation of CYP11A transcription.