©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Two Sp1-binding Sites Mediate cAMP-induced Transcription of the Bovine CYP11A Gene through the Protein Kinase A Signaling Pathway (*)

(Received for publication, June 5, 1995; and in revised form, August 21, 1995)

Pratap Venepally (§) Michael R. Waterman (¶)

From the Department of Biochemistry, Vanderbilt University, School of Medicine, Nashville, Tennessee 37232-0146

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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.


MATERIALS AND METHODS

Oligonucleotides

Complimentary single-stranded oligonucleotides were synthesized and annealed to generate the following double-stranded oligonucleotides representing both wild type and mutant bovine CYP11A CRS fragments. The sequence for a consensus GC-rich double-stranded oligonucleotide that binds transcription factor Sp1 is also shown (k). The staggered sequences, in lower case letters, at the 5`- and 3`-ends of the antisense strand were included to enable the insertion of the oligonucleotides into SacI and SalI restriction enzyme sites of OVEC or luciferase reporter plasmid DNA. The bold letters identify the positions where changes in wild type sequence were made to create mutant oligonucleotides. (a) -118/-100, 5`-cgagACTGAGTCTGGGAGGAGCTg-3`, 3`-tcgagctcTGACTCAGACCCTCCTCGAcagct-5`; (b) -116/-114M, 5`-cgagACATTGTCTGGGAGGAGCTg-3`, 3`-tcgagctcTGTAACAGACCCTCCTCGAcagct-5`; (c) -108/-107M, 5`-cgagACTGAGTCTGCCAGGAGCTg-3`, 3`-tcgagctcTGACTCAGACGGTCCTCGAcagct-5`; (d) -105/-104M, 5`-cgagACTGAGTCTGGGACCAGCTg-3`, 3`-tcgagctcTGACTCAGACCCTGGTCGAcagct-5`; (e) -103/-101M, 5`-cgagACTGAGTCTGGGAGGTCATg-3`, 3`-tcgagctcTGACTCAGACCCTCCAGTAcagct-5`; (f) -118/-104, 5`-cgagACTGAGTCTGGGAGGg-3`, 3`-tcgagctcTGACTCAGACCCTCCcagct-5`; (g) -111/-100, 5`-cgagCTGGGAGGAGCTg-3`, 3`-tcgagctcGACCCTCCTCGAcagct-5`; (h) -101/-50, 5`-cgagCTGTGTGGGCTGGAGTCAGCCGGAGGAGGCTGACCGCCCTGTCAGCTTCTCAG-3`, 3`-tcgagctcGACACACCCGACCTCAGTCGGCCTCCTCCGACTGGCGGGACAGTCGAAGAGTCagct-5`; (i) -70/-32, 5`-cgagGACCGCCCTGTCAGCTTCTCACTTAGCCTTGAGCTGGTGG-3`, 3`-tcgagctcCTGGCGGGACAGTCGAAGAGTGAATCGGAACTCGACCACCagct-5`; (j) -70/-50, 5`-cgagGACCGCCCTGTCAGCTTCTCAg-3`, 3`-tcgagctcCTGGCGGGACAGTCGAAGAGTcagct-5`; (k) consensus Sp1 oligonucleotide, 5`-CCTCGAGATCGGGGCGGGGCGATG-3`, 3`-tcgaGGAGCTCTAGCCCCGCCCCGCTACagct-5`.

Plasmid Construction

The beta-globin reporter gene plasmids, OVEC, OVECREF, and SVOVEC (Westin et al., 1987), were obtained from Drs. Thomas Gerster and Walter Schaffner, University of Zurich, Switzerland. The primary vector OVEC contains a rabbit beta-globin coding sequence and a minimal beta-globin promoter and is used in the subcloning of promoter fragments. The luciferase reporter gene plasmids, pA(3)LUC, a promoterless vector (Maxwell et al., 1989; Wood et al., 1989) and pRSV186LUC, which contains the enhancerless Rous sarcoma virus (RSV) promoter, were provided by Dr. David Gordon (University of Colorado). The vector pA(3)LLUC was constructed by inserting a 100-bp KpnI/HindIII polylinker fragment from the PHECATN plasmid (5 Prime 3 Prime, Inc., Boulder, CO) into the unique KpnI/HindIII sites of pA(3)LUC.

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 times and 2 times -126/-113h21OV (Kagawa and Waterman, 1992), 1 times and 2 times -243/-225b17OV (Lund et al., 1990), and 699/740ADXOV (Chen and Waterman, 1992), respectively. The plasmid 4 times CREOV (Lund et al., 1990) contains four tandem copies of the CRE from the human chorionic gonadotropin-alpha 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 beta-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-beta-globin reporter constructs. The CYP11A promoter sequences shown in B were inserted between SacI (ScI) and SalI (SlI) sites in front of a minimal beta-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(3)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 pA(3)LLUC 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, CEValphaNeo, and Mt-REV(AB)-Neo, respectively, were kindly provided by Dr. Stanley McKnight, University of Washington, Seattle.

Preparation of Nuclear Extracts

Nuclear extracts were prepared from mouse Y1 adrenocortical cells as described by Dignam et al.(1983) and simplified for small scale preparation as described by Andrews and Faller(1991). Protein concentrations were determined by the BCA assay (Pierce Chemical Co., Rockford, IL) using bovine serum albumin as standard.

Gel Shift Assay

The double-stranded oligonucleotides were labeled by polynucleotide kinase and [-P]dATP (6,000 Ci/mmol, Amersham Corp.) or by a fill-in reaction with [alpha-P]dCTP (3,000 Ci/mmol, Amersham Corp.) and DNA polymerase I, Klenow fragment. Twenty-five µg of nuclear extract protein or 7.5 ng of purified Sp1 transcription factor (Promega Biotech Inc.) were mixed with 15 µl of binding buffer (20 mM Hepes, pH 7.9, 80 mM KCl, 5 mM MgCl(2), 2% Ficoll, 5% glycerol, 0.1 mM EDTA, 0.2 mM DTT), 2 µg of poly(dI-dC), and P-labeled probe (10,000 cpm) on ice. For competition assay, 10 pmol of unlabeled competitor oligonucleotide was used along with the labeled probe. In the case of antibody supershift experiments, the incubation conditions were identical except 1-2 µg of anti-Sp1 antibody (Santa Cruz Biotech., Santa Cruz, CA) was preincubated with 25 µg of nuclear extract proteins from Y1 cells in the presence of binding buffer for 10 min prior to the addition of other components. The DNA-protein complexes were resolved by electrophoresis on a 5% polyacrylamide, 0.5% Ficoll gel (Sambrook et al., 1989) and visualized by autoradiography.

Cell Culture and Transient DNA Transfections

The Y1 and KIN8 cells were routinely maintained in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum at 37 °C in a 5% CO(2) incubator. Near confluent cell monolayers were trypsinized and split (1:6) into 10-cm plates 24 h before the addition of DNA precipitates. Fresh medium was added to the cells 4 h before transfection. Nineteen µg of test plasmid plus 1 µg of internal reference vector, OVECREF (in the case of beta-globin reporter assays), or 15 µg of test plasmid together with 4 µg of CEValphaNeo/Mt-REV(AB)-Neo, and 1 µg of OVECREF (in the case of PKA cotransfection experiments) were precipitated with 0.125 mM CaCl(2) in the presence of 50 mM Hepes, 1.5 mM sodium phosphate, 280 mM NaCl, pH 7.12 (Graham and Van Der Eb, 1973; Gorman et al., 1982). One ml of DNA-calcium phosphate precipitate was added to each plate of cells. After 4 h of exposure to the DNA precipitates, the cells were shocked with 15% glycerol for 3 min at room temperature. The medium was changed the morning after transfection, and the cells were maintained in the presence of either 25 µM forskolin or dimethyl sulfoxide (vehicle) for 6-8 h. The medium used after PKA subunit transfections always contained 100 µM ZnSO(4) to activate metallothionein promoter-controlled PKA subunit expression. For luciferase reporter gene assays the transfection procedure was the same as described before except 20 µg of test plasmid were used, and the cells were exposed to forskolin for 36 h after the treatment with glycerol.

RNA Isolation and S1 Nuclease Analysis

Two alternate procedures were followed for the preparation of RNA from transfected cells. In the first method, the RNA was prepared as described by Lund et al.(1990). In the second, RNA was isolated using a commercially available Ultraspec reagent (Biotecx Laboratories, Inc., Houston, TX). After removal of medium, 2 ml of Ultraspec reagent were added directly to the cells on the plate for lysis. The lysate was mixed by vortexing, transferred into 15-ml tubes, and kept on ice for 5 min. Following the addition of 400 µl of chloroform to the lysate, the samples were mixed vigorously and incubated on ice for an additional period of 5 min. After a 10-min centrifugation at 10,000 rpm, the RNA in the supernatant was precipitated with an equal volume of isopropanol. S1 nuclease analysis on 10-50 µg of cytoplasmic RNA was performed as described previously (Lund et al., 1990). After autoradiography, the intensities of the bands corresponding to the correctly initiated transcripts were quantitated using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

Luciferase Assays

Thirty-six hours after the treatment with glycerol, the cells were lysed with 1 ml of 1% Triton X-100, 25 mM glycylglycine, pH 7.8, 15 mM MgSO(4), 4 mM EGTA, and 1 mM DTT. The assays were performed essentially as described previously by Brasier et al.(1989) and Venepally et al.(1992). One hundred µl of lysate were added to 360 µl of 25 mM glycylglycine, pH 7.8, 15 mM MgSO(4), 4 mM EGTA, 15 mM potassium phosphate, pH 7.8, 1 mM DTT, and 2 mM ATP. The luciferase reactions were initiated by the injection of 100 µl of 25 mM glycylglycine, pH 7.8, 15 mM MgSO(4), 4 mM EGTA, 2 mM DTT, and 0.2 mM luciferin into the sample, and light output was measured for 20 s at 25 °C using a monolight luminometer (Analytical Luminescence Laboratory, San Diego, CA).


RESULTS

Mutations in the -118/-100 Sequence Affect Formation of DNA-Sp1 Complexes

The proximal promoter region of the bovine CYP11A gene (Fig. 1A) that supports cAMP-induced transcription in Y1 adrenocortical cells (Ahlgren et al., 1990) contains potential binding sites for the general transcription factor Sp1 (Kadonaga et al., 1986), steroidogenic factor, SF-1 (Morohashi et al., 1992; Lala et al., 1992), and adrenal specific factor, ASP (Kagawa and Waterman, 1992). Deletion analyses of this region have previously shown that the sequence between -118 and -100 is sufficient for supporting cAMP-dependent transcription in both Y1 (Momoi et al., 1992) and bovine ovarian luteal cells (Begeot et al., 1993). Moreover, the same sequence with nuclear extracts from bovine ovarian luteal cells (Begeot et al., 1993) and a slightly larger fragment, extending from -118 to -83, with nuclear extracts from Y1 cells show binding to proteins that also interact with the consensus Sp1 binding sequence. To more precisely identify the nucleotides important for the binding of these nuclear proteins, several mutations were made within the -118/-100 sequence (Fig. 1B) and their effects on the DNA-protein interactions were examined using Y1 nuclear extracts. As seen in Fig. 2A, both wild type -118/-100 and a consensus Sp1 binding sequence produce similar complexes I, II, and III, although the latter showed greater affinity. Only the -116/-114M sequence and the consensus Sp1 oligonucleotide competed for the formation of these complexes by the wild type -118/-100 sequence.


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 (alpha-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.

Transient Expression Analysis of Wild Type and Mutant CYP11A Reporter Gene Plamids in Y1 Cells

To examine the effects of mutations and deletions in the -118/-100 region on cAMP-dependent transcription, the wild type and mutant -118/-100 sequences, used in in DNA-protein interaction analyses shown in Fig. 2, were inserted into OVEC-beta-globin reporter gene vectors (Fig. 1C) and transfected into Y1 adrenal tumor cells. Cytoplasmic RNA, isolated from cells treated with and without (control) forskolin, was analyzed by S1-nuclease digestion for expression of the beta-globin reporter mRNA. As seen in Fig. 3A, forskolin treatment resulted in a 5-6-fold increase over control levels of beta-globin RNA expression with both the -118/-100OV wild type and -116/-114MOV mutant plasmids. The fold-induction in response to forskolin observed with these two constructs is similar to that obtained with -896/-32OV, although, the latter supported the highest level of activity. Promoter constructs with mutations between -108 and -100 bp, namely -103/-101MOV, -105/-103MOV, and -108/-107MOV, showed a markedly decreased forskolininduced transcription compared with either -118/-100OV or -116/-114MOV. Both basal and forskolin-induced transcription observed from these mutant constructs was comparable to that seen with the empty vector (OVEC). Also, the -111/-100OV plasmid was as efficient as the longer wild type -118/-100 construct in supporting cAMP (forskolin)-induced transcription in Y1 adrenocortical tumor cells (Fig. 3B). Thus, the mutations in the G-rich region of the -118/-100 sequence that reduced or eliminated the formation of DNA-protein complexes I and II in Y1 nuclear extracts (Fig. 2B) also markedly reduced cAMP-induced transcription in Y1 cells.


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(4) 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 beta-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.



Analysis of the Sequences between -100 and -32 bp

The data from the initial deletion analysis of the 5`-flanking region of bovine CYP11A (Ahlgren et al., 1990) showed that, although the fragment from -186/-32 promoted near-maximal cAMP-dependent transcription, a smaller sequence between -101 and 32 retained the ability to promote a 9-fold increase in cAMP-dependent transcription over control levels in Y1 cells. This suggested the possibility that more than one cAMP-responsive sequence might be present in the proximal 5`-flanking region of the bovine CYP11A promoter. To look for elements in addition to -111/-100, two overlapping fragments, -101/-50 and -70/-32 spanning the sequence -101 to -32 bp, were screened for their ability to bind to nuclear proteins from Y1 cells. As seen in Fig. 4A, both overlapping fragments -101/-50 and -70/-32 formed a complex similar to complex I observed with the -111/-100. Formation of this complex disappeared when the unlabeled consensus Sp1 oligonucleotide was used as a competitor. Complexes III and IV, observed in the case of -101/-50 and -70/-50, respectively, were not affected by such competition. Further, in each case, protein(s) present in complex I interacted with the Sp1 antibody, resulting in a supershift complex, suggesting that, as with -111/-100, either Sp1 or proteins antigenically related to it bind to sequences within -101/-50 and -70/-32. A sequence that shares homology with the consensus Sp1-binding site is present between -70 and -60 of the CYP11A promoter (Fig. 1A). Thus, the -70/-50 sequence, which is common to fragments -101/-50 and -70/-32, was compared with the -118/-100 sequence for both its ability to bind Sp1 and to support cAMP-induced transcription in Y1 cells (Fig. 4, B and C). In the presence of Y1 nuclear extracts, both fragments -118/-100 and -70/-50 yielded complexes I and II, although the former showed stronger complex formation (Fig. 4B). Also, from the appearance of a supershift complex in the presence of Sp1 antibody, the complexes formed by -70/-50 appear to contain Sp1 or a protein antigenically related to it. Addition of antibody against CREB transcription factor did not affect any of the complexes, revealing that CREB is not involved in their formation. Moreover, when assayed by transient expression analysis, the plasmids containing both -118/-100 and -70/-50 fragments supported similar levels of cAMP-induced transcription in Y1 cells (Fig. 4C). Thus, there is an additional cAMP-responsive element in the bovine CYP11A promoter region which is located between -70 and -50 bp, and this sequence like -111/-100 has the ability to bind Sp1. Consistent with these results, when -186/+12 and -101/+12 CYP11A fragments, which contain both or only the proximal Sp1-binding site(s), respectively, were analyzed for transcriptional activity in Y1 cells (Fig. 4D), the former showed a 5-fold greater activity than the latter in both basal and cAMP-induced transcription. However, as compared to the vector A(3)LLUC, the -101/+12 fragment itself supported a 10- and 20-fold increase in basal and cAMP-induced transcription, respectively. Thus, the transcriptional elements present between -186 and -101 and between -101 and +12 of the bovine CYP11A promoter each participate in both basal and cAMP-induced transcription in Y1 cells.


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 alpha-Sp1, anti-Sp1 antibody; alpha-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.



cAMP-induced Transcription Supported by -118/-100 and -70/-50 Sequences Is Mediated by Protein Kinase A

The role of PKA in the forskolin-stimulated transcription supported by CYP11A sequences was examined by the analysis of reporter gene expression in KIN8 and Y1 cells (Fig. 5). When the activity of promoter constructs containing one or two copies of the -118/-100 sequence was analyzed in PKA-deficient KIN8 cells, little cAMP-induced transcription was observed over the background levels (Fig. 5A). However, the same plasmids showed a 5-6-fold increase in the reporter gene transcription when the free catalytic subunit of PKA was overexpressed in these cells. As expected, forskolin treatment did not enhance the PKA catalytic subunit-dependent transcription, since the catalytic subunit does not interact with cAMP (Rae et al., 1979). The high level of transcriptional activity observed from the SV40OV was unaffected by the absence or the presence of a functional PKA.


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(4) 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 beta-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 times 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 times CREOV, the plasmid that contains two consensus CRE elements, showed a very high level of transcriptional activity under these conditions.

Comparison of cAMP Responsiveness of Various CRS Elements Involved in Steroidogenesis

The levels of basal and cAMP-dependent transcription supported by the -118/-100 and -70/-50 fragments of the bovine CYP11A gene and CRSs characterized in other genes involved in steroidogenesis were compared to evaluate their relative strengths (Fig. 6). An approximate 3-5-fold increase in cAMP-dependent transcription was observed by equivalent constructs containing one or two tandem copies of bovine CYP11A CRSs (-118/-100 and -70/-50), bovine CYP17 CRS1 (-243/-225) and human CYP21 CRS (-126/-113) promoter sequences. The CRS from intron 1 of the bovine adrenodoxin gene in ADXOV (699/740) (Chen and Waterman, 1992) that contains two GC-rich sequences supported the highest level of basal and cAMP-induced transcription among the genes associated with steroidogenic metabolism. The level of cAMP-induced expression directed by 2 times CREOV was similar to that observed with 1 times ADXOV, although the induction effect observed in the case of the former was much higher because of its low basal activity. OVEC served as the control for background expression.


Figure 6: Comparison of transcriptional activity supported by CRSs of different genes involved in steroidogenesis. Transcriptional activity directed by promoter constructs containing one (1times) or two (2times) 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.




DISCUSSION

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 IIbeta 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 beta-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.


FOOTNOTES

*
These studies were supported by United States Public Health Service Grant DK-28350. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Pharmaceutical Division, Biological Sciences KMI-112, Southern Research Institute, 2000 9th Ave. South, Birmingham, AL 35299.

To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University, School of Medicine, 607 Light Hall, Nashville, TN 37232-0146. Tel.: 615-322-3318; Fax: 615-322-4349.

(^1)
The abbreviations used are: bp, base pair(s); CRS, cAMP-response sequence; PKA, cAMP-dependent protein kinase; CRE, cAMP-response element (TGACGTCA); DTT, dithiothreitol; RSV, Rous sarcoma virus.


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