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Endocrinology Vol. 145, No. 2 901-912
Copyright © 2004 by The Endocrine Society

Transcriptional Regulation of P450scc Gene Expression in the Embryonic Rodent Nervous System

Fabian Hammer, Nathalie A. Compagnone, Jean-Louis Vigne, Susanna R. Bair and Synthia H. Mellon

Department of Obstetrics, Gynecology, and Reproductive Sciences, The Center for Reproductive Sciences, and The Metabolic Research Unit, University of California, San Francisco, San Francisco, California 94143

Address all correspondence and requests for reprints to: Synthia H. Mellon, Ph.D., Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, 513 Parnassus Avenue, Box 0556, San Francisco, California 94143-0556. E-mail: mellon{at}cgl.ucsf.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Steroid hormones are synthesized in adrenals, gonads, placenta, and the central and peripheral nervous systems (neurosteroids). Neurosteroidogenesis, like conventional steroidogenesis, begins with the conversion of cholesterol to pregnenolone, catalyzed by mitochondrial P450 side-chain cleavage enzyme (P450scc). Transcription of the P450scc gene in the adrenals and gonads requires steroidogenic factor-1, which is not expressed in the nervous system cells that express P450scc. A crucial transcriptional regulatory region of the rat P450scc gene is at -130/-94. We have purified two nuclear proteins (70 and 86 kDa) from rat glial C6 cells that specifically bind to the -130/-94 region of the rat P450scc promoter and identified them as the DNA-binding subunits of autoimmune antigen Ku. Ku colocalized with P450scc in several regions of the nervous system, but its overexpression in C6 cells did not augment transcription from a -130/-94 Luciferase construct. Members of the Sp family of transcription factors also bind to the same DNA sequence as Ku. Sp4 and Sp2 colocalize with P450scc in the nervous system early in development, whereas Sp1 and Sp4 colocalize later in development. Sp1 robustly increased transcription from this element in Sp-deficient Drosophila SL2 cells, and Ku synergistically enhanced this Sp1-stimulated transcription. Thus, members of the Sp transcription family play a role in activating P450scc gene transcription in the nervous system, and Ku may further augment this activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEROID HORMONES ARE synthesized in the central and peripheral nervous systems as well as in gonads, adrenals, and placentae (reviewed in Ref. 1). The conversion of cholesterol to neuroactive steroids (neurosteroids) in the nervous system uses many of the same steroidogenic enzymes used in adrenal and gonadal steroidogenesis (2, 3, 4, 5, 6). Some neurosteroids are the same as those synthesized in classic steroidogenic tissues, and some are distinct, but neurosteroids do not act on classic nuclear steroid hormone receptors; rather, they modulate neuronal activity by binding to and activating neurotransmitter receptors such as {gamma}-aminobutyric acid type A and N-methyl-D-aspartate on the cell surface (7, 8, 9, 10). Neurosteroids include 3{alpha}5{alpha} derivatives of progesterone; deoxycorticosterone; and testosterone, called allopregnanolone; tetrahydrodeoxycorticosterone; and androstanediol, respectively, as well as the 17{alpha}-hydroxylated C19 derivatives of pregnenolone, dehydroepiandrosterone (DHEA), and its sulfated ester, DHEAS. Allopregnanolone acts as an endogenous anxiolytic, anesthetic, anticonvulsant compound by increasing the frequency and duration of {gamma}-aminobutyric acid type A channel opening (reviewed in Ref. 11). Allopregnanolone may also cause regression of neurites from hippocampal neuronal cultures, suggesting it has additional functions (12). DHEA and DHEAS are involved in neuronal modeling because they stimulate growth of embryonic axons (DHEA) and dendrites (DHEAS) (13).

Because only one enzyme, P450 side-chain cleavage (P450scc), initiates steroidogenesis in both the nervous system and classic steroidogenic tissues, we sought to determine whether the single gene for P450scc was under basal regulation by the same nuclear factors in classic steroidogenic tissues and the nervous system. Previous studies identified a region -54/-35 bp upstream from the transcriptional start site of the rat P450scc gene to which the orphan nuclear receptor steroidogenic factor (SF-1) binds and which is required for ovarian (14), testicular (15), and adrenal expression (15). Although SF-1 is expressed in the brain (16, 17) and may colocalize with P450scc in the pituitary (2, 3), SF-1 expression was restricted to the pituitary and ventromedial hypothalamus and hence was not found in most brain regions that express P450scc (3). Rat C6 glial cells express the P450scc gene but lack SF-1 and contain no other factor that binds to this -54/-35 region but instead have a factor that binds at -130/-94 (15). We have now purified this factor from C6 cells and identified it as the p70 and p86 subunits of Ku autoimmune antigen. We have also identified members of the Sp family of transcription factors that also bind to this same region and colocalize with P450scc at different times in development. These factors may be required for the developmentally regulated basal expression of P450scc in the rodent nervous system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture, transfections, and luciferase assays
Rat glial C6 cells obtained from the American Type Culture Collection (Manassas, VA) were grown in DMEM containing 4.5 mg glucose/ml and 10% fetal bovine serum. All cells were plated at 200,000 cells per 35-mm well on the day before transfections. Transfection was performed with a lipofection reagent (FuGene; Roche Applied Science, Indianapolis, IN), using 1 µg DNA per well. When vectors expressing Ku were cotransfected with luciferase reporter constructs, the molar ratio of the plasmids was 1:1. DNA concentrations were equalized by the addition of the cloning vector pCR3.

Drosophila Schneider’s SL2 cells were cultured in Schneider’s Drosophila medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, L-glutamine, and penicillin/streptomycin at 27 C. Cells were plated at 2 x 106 cells per 35-mm well the day before transfections. Transfections were performed using a nonliposomal lipid transfection reagent (Effectene; Qiagen, Valencia, CA) according to the manufacturer’s protocol, using 200 ng DNA of the reporter construct per well. When vectors expressing Sp1 were cotransfected with luciferase reporter constructs, 100 ng Sp1 DNA expression plasmid (pPac-Sp1) or vector alone (pPac-0) was used. DNA concentrations were equalized by the addition of cloning vector pPac-0. For expression of Ku, 200 ng of the p70- and p80-expression plasmid (CMV-Ku70, CMV-Ku80) were cotransfected with the luciferase reporter and Sp1 expression plasmid. The Drosophila Sp1 expression vectors pPac-Sp1 and pPac-0 were generous gifts from Dr. G. Suske (Institut für Molekularbiologie und Tumorforschung, University of Marburg, Marburg, Germany). Luciferase assays were performed after 48 h.

Luciferase assays and data analysis were as described elsewhere (18), using a Monolight 1500 luminometer (Analytical Luminescence Laboratory, San Diego, CA) and a luciferase assay system (Promega, Madison, WI). Protein concentrations were assayed using the BCA protein assay kit (Pierce, Rockford, IL).

Gel shift assays
Nuclear protein extracts from C6 cells were prepared as previously described (19, 20). Gel shift assays were performed as described (15, 19, 21, 22, 23). A synthetic oligonucleotide, containing the DNA sequences between -94 and -130 of the rat P450scc gene was used in mobility shift assays and also cloned into luciferase reporter gene vectors for use in transient transfection assays. Oligonucleotide probes were end labeled using [{gamma}-32P]ATP and T4 polynucleotide kinase, mixed with the nuclear proteins in the presence of 100 µg/ml poly (dI/dC), 50 µg/ml salmon sperm DNA, 5 mM dithiothreitol (DTT), and 1 mg/ml BSA and binding buffer [20 mM HEPES (pH 7.9), 60 mM KCl, 4 mM Tris HCl (pH 7.9), 600 µM EDTA, and 600 µM EGTA] and incubated at room temperature for 40 min.

To diminish nonspecific binding of Ku to DNA, the binding buffer included 200 µM MgCl2, 200 µM ZnCl2, and 11% glycerol. Nuclear protein, buffer, poly dI/dC, salmon sperm, DTT, and BSA (i.e. all components except for the labeled probe) were preincubated at room temperature for 15 min before addition of the 32P-labeled probe (24). A quarter of the total reaction was loaded onto a 6% nondenaturing polyacrylamide gel, using 0.5x Tris-boric acid-EDTA as running buffer, to separate the free labeled probe from probe bound by nuclear protein (1x Tris-boric acid-EDTA is 89 mM Tris, 89 mM boric acid, and 2 mM EDTA). The dried gel was then exposed to x-ray film.

Purification of proteins bound to the -130/-94 region of the rat P450scc gene
C6 cells were grown in roller bottles to obtain large (>10 ml) cell pellets. Nuclei were prepared by resuspending the cell pellet in 10 ml buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN). Cells were incubated on ice for 30 min, and swollen cells were disrupted using a Dounce homogenizer with a B-pestle. Cells were homogenized until more than 95% of the cells were lysed, judged by light microscopy. The homogenate was centrifuged for 10 min at 3,000 x g to pellet the nuclei, and the nuclear pellet was resuspended in 2 volumes of 20 mM HEPES (pH 7.9), 10 mM KCl, 20% glycerol 1 mM DTT, 1 mM PMSF, and 0.2% Nonidet P-40 (Sigma, St. Louis, MO), and nuclear extracts were gently shaken at 4 C for 30 min. Nuclear membranes were pelleted by centrifugation of the nuclear extracts at 16,000 x g for 30 min, and the remaining supernatant containing nuclear proteins was dialyzed overnight at 4 C against 20 mM HEPES (pH 7.9), 100 mM KCl, 20% glycerol, 1 mM DTT, and 1 mM PMSF.

Nuclear proteins were precipitated by addition of various concentrations of ammonium sulfate; the proteins that bound to the -130/-94 P450scc oligonucleotide precipitated in the 40–60% ammonium sulfate cut. These proteins were dialyzed against 20 mM Tris-Cl (pH 7.5), centrifuged for 1 h at 100,000 x g, and proteins remaining in the supernatant were separated by fast performance liquid chromatography (FPLC) on a Mono Q anion exchange column, previously equilibrated in 20 mM Tris-Cl (pH 7.5). Fractions eluted from the Mono-Q column (8 ml Protein Pack Q 8HR FPLC Anion Exchange column; Waters, Milford, MA) were monitored for protein by absorbance at 280 nm. Proteins not retained on the column were eluted in Tris-Cl buffer until the absorbance at 280 nm returned to zero. Proteins retained on the column were eluted with a linear gradient of 0–1 M NaCl, generated by a Waters controller system, model 600E, at a flow rate of 1 ml/min. Proteins eluting from the column were monitored for binding to the -130/-94 P450scc oligonucleotide by gel shift assays of aliquots of each sample. Most of the proteins that eluted from the MonoQ column were found in fractions 15–24 and fractions 44–49. By gel shift analysis, the protein binding to the -130/-94 P450scc oligonucleotide was found in fractions 29–31. Another protein binding to -130/-94 was found in fractions 21–25; this protein was never identified in the original C6 extract, and its relevance is uncertain.

Protein sequencing
Proteins displayed on a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel were transferred to a polyvinyl difluoride (PVDF) membrane, (Bio-Rad Laboratories, Richmond, CA) in 10 mM 3-[cyclohexylamino]-1-propanesulfonic acid buffer (pH 11), in 10% methanol. Transferred proteins were stained in 0.1% Coomassie Brilliant Blue R-250 in 50% methanol/1% acetic acid and were destained in 50% methanol. The bands of interest were excised from the PVDF membrane and sent for N-terminal microsequencing (ProSeq, Inc., Salem, MA). Sequences obtained were searched for homology with sequences in the SWISS-PROT database, using the FASTA search of the GCG program.

Western blots
Proteins separated on 10% SDS polyacrylamide gels were transferred to nitrocellulose membranes. Blots were subjected to Western analysis using polyclonal antibodies raised against human Ku 70 and Ku 86 peptides (Santa Cruz Biotechnology, Santa Cruz, CA).

Immunocytochemistry
All animal experimentation was performed in accordance with protocols and guidelines approved by the Committee for Animal Research at the University of California, San Francisco. For immunocytochemical studies, mouse embryos from embryonic d 9.5, 10.5, 17.5, and 18.5 were screened. Immunocytochemistry on fresh frozen tissues was performed as described (3, 4) using antibodies against human Ku 70, Ku 86, Sp1, Sp2, Sp3, and Sp4 peptides (Santa Cruz Biotechnology) and recombinant human P450scc (25) and using a fluorescein isothiocyanate (FITC)-conjugated second antibody. For control experiments, primary antibodies were immunoabsorbed with peptides used to generate the antibodies. Because the antibodies to P450scc and Sp were generated in rabbits, we biotinylated antibodies to colocalize two or more antibody-antigen complexes on a single tissue section, using the manufacturer’s procedures (Molecular Probes, Inc., Eugene, OR). Sp antibodies were concentrated from 0.5 ml (200 mg/ml) to 0.2 ml (0.5 mg/ml) using a spin column (Bio-Rad Laboratories), and 1/10 volume (20 µl) of 1 M sodium bicarbonate was added. The solution was stirred, and 2 µl biotin-sulfosuccinimidyl-ester, resuspended in 200 µl distilled water, was added. The reaction was stirred for 1.5 h and the free biotin was separated from the biotin incorporated into the protein by centrifugation at 1100 x g for 5 min in a spinning bucket rotor using the column provided by the manufacturer (Molecular Probes, Inc.).

For double immunostaining with Ku antibodies, slides were fixed for 20 min with 4% paraformaldehyde in PBS, washed three times for 5 min in PBS and blocked with PBS containing 10% fetal calf serum and 0.3% TritonX-100. After blocking, slides were incubated for 2 h with the p70 or p86 Ku antibody. The slides were washed with PBS three times for 5 min each, incubated with antigoat rhodamine antibody (1:200 dilution in PBS), and excess antibody washed off in PBS (three times, 5 min each). Slides were then incubated with PBS containing 10% goat serum to block the secondary antigoat antibody and then incubated with P450scc primary antibody at a 1:500 dilution. The slides were washed in PBS (three times, 5 min each time), incubated with the secondary antirabbit-FITC antibody (1:200 dilution in PBS) for 30 min, and secondary antibody was washed in PBS (three times, 5 min each).

For double immunostaining with Sp antibodies, slides were incubated with PBS containing 10% rabbit serum to block the secondary antirabbit-FITC antibody. After blocking the slide the second time, it was incubated with the biotinylated Sp antibody (1:100 dilution in PBS) for 2 h, washed three times with PBS for 5 min, and incubated with Extravidin-Cy3 (Sigma) (1:200 dilution in PBS) for 30 min. Extravidin not bound to the biotinylated antibody was washed off the slide in PBS (three times, 5 min each). Slides were mounted in 50% PBS/50% glycerol.

For triple immunostaining with Sp1, Sp4, and P450scc antibodies, we labeled the third antibody with a rabbit IgG to which a fluorescent probe, Alexa Fluor 350, was attached (Zenon rabbit IgG labeling kit), according to the manufacturer’s protocol (Molecular Probes). Nonspecific sites were blocked using rabbit IgG supplied by the vendor. Slides previously doubly immunostained as described above were incubated with in PBS containing 0.2% Triton X-100 (PBT) for 20 min at room temperature. Nonspecific binding sites were blocked with PBT containing 5% normal rabbit serum for 30 min at room temperature. After blocking the slide the third time, it was incubated with the Alexa-fluor 350-labeled antibody (1:100 dilution in PBT) for 1 h, washed three times with PBT for 10 min, three times with PBS for 10 min, and fixed a second time in 4% paraformaldehyde in PBS for 15 min at room temperature. The slides were then washed three more times with PBS and mounted in 50% glycerol:50% PBS.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Purification of the C6 nuclear factor that binds to the -130/-94 region of the rat P450scc gene
We have shown that the rat P450scc gene is transcriptionally active in rat C6 glial cells (15) and that a basal element required for expression in C6 cells was located between 130 and 94 bp upstream from the transcriptional start. Although this region was also active in mouse Leydig MA-10 cells, the basal element required for activity in MA-10 cells was between the TATA box and -94 and could be bound by the transcription factor SF-1.

A single protein appeared to bind to the -130/-94 region (Fig. 1AGo). We purified this protein from C6 glioma cells, using ammonium sulfate precipitation of nuclear proteins, followed by FPLC chromatography. The 40–60% ammonium sulfate fraction contained DNA binding activity and was purified further by FPLC (Fig. 1Go). Gel shift analysis of the fractions from this column showed most DNA binding activity in fractions 29–31 (Fig. 1BGo). The protein(s) binding to DNA found in fractions 19–25 had not been observed previously and was not studied further. SDS gel electrophoresis and silver staining of fractions 29–31 showed that two proteins of about 70 and 80 kDa were especially enriched in fractions containing DNA binding activity (Fig. 1CGo). These protein bands were transferred to PVDF membranes, and the 80 kDa protein was extracted and sequenced by Edman degradation. About 40–50 pmol of protein yielded the sequence of 19 amino acids (Fig. 1DGo) and Basic Local Alignment Search Tool analysis of the Swiss protein database identified this sequence as amino acids 2–20 of the 86,000 subunit of the multimeric protein Ku (Fig. 1DGo).



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  		FIG. 1. Analysis of purified proteins from C6 cells. A, Autoradiogram of a gel shift analysis of the -130/-94 region of the rat P450scc gene. Extracts from C6 cells were incubated with a probe from the -130/-94 region of the rat P450scc gene. The single protein/DNA complex formed with C6 extracts is competed by 100-fold molar excess of unlabeled -130/-94 rP450scc oligonucleotide (+cold -130/-94). B, Gel shift analysis of fractions from the Mono-Q FPLC. Two-microliter aliquots from each fraction from the Mono-Q FPLC column were combined with the -130/-94 rP450scc probe, and protein/DNA fractions were separated on SDS-polyacrylamide gels. A protein/DNA complex similar to that seen with the starting material (lane SM) was found in fractions 29–31, indicated by the arrow on the right. C, Silver-stained SDS-polyacrylamide gel of aliquots of fractions from a Mono-Q FPLC, containing proteins purified from C6 cells. Arrows indicate two proteins of about 70 and 80 kDa that are enriched in fractions 29–31, corresponding to the fractions that contain the protein/DNA complex seen in A. D, Partial amino acid sequence of the 80-kDa protein isolated from C6 cells. The proteins obtained from C6 cells in fraction 29 (C) were transferred to PVDF membranes, and the 80-kDa protein was sequenced by sequential Edman degradation. The 19-amino acid sequence obtained was analyzed and compared with other protein sequences in the Swiss protein database using Basic Local Alignment Search Tool analysis (BLAST), which found a 100% match with amino acids 2–20 of the 86-kDa subunit of the human autoantigen Ku.

 
Ku is composed of two subunits, p70 and p86, that must form heterodimers to bind DNA. Western blots of protein fractions from the Mono-Q FPLC column using antibodies against the p70 Ku subunit detected this Ku subunit in fractions 23–37, with the greatest concentration of this protein in fraction 29 (Fig. 2AGo). Similarly, antibodies against the p86 subunit of Ku detected this Ku subunit in fractions 27–35, with the greatest concentration in fraction 29. Thus, the p70 and p86 subunits of Ku copurify on the final FPLC column, and the fractions containing the protein/DNA complex seen on gel shift assays (fractions 29–31, Fig. 1BGo) also contain the highest concentration of the p70 and p86 Ku subunits, indicating that these proteins are those found in the protein/DNA complex.



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  		FIG. 2. Analysis of Ku in proteins purified from C6 cells. A, Western blot analysis of fractions from the MonoQ FPLC. Aliquots of proteins obtained from the MonoQ purification of C6 cells were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with antibodies against the p70 (top) or p86 (bottom) subunit of human Ku. The p70 subunit was found in fractions 23–37, and p86 subunit was found in fractions 27–35. B, Gel shift analysis of C6 cell proteins. Aliquots of C6 cell proteins were incubated with the -130/-94 rP450scc oligonucleotide probe in the absence or presence of antibody against the p70/p86 Ku dimer. The protein/DNA complex was completely shifted with this antibody but not with nonimmune rabbit serum (NRS), indicating that both proteins were found in this complex and that the complex was due to Ku dimer binding.

 
The Ku p70 and p86 proteins bind as a complex to the -130/-94 region of the P450scc gene
To determine whether the protein from C6 cells that bound to the -130/-94 region of the P450scc gene was indeed Ku, we performed gel shift analyses with monoclonal antibodies to the Ku p70/p86 dimer (26). Antibodies to the p70/p86 dimer could supershift the entire protein/DNA complex (Fig. 2BGo), further indicating that the protein/DNA complex was due solely to the Ku p70/p86 dimer binding to DNA.

Colocalization of Ku p70 and Ku p86 with P450scc in the nervous system
Immunocytochemical analysis of sections of embryonic d 18.5 mice showed that p70 and p86 are coexpressed in several regions of the brain, and these regions correspond to those in which we previously detected P450scc. Immunocytochemistry for P450scc and Ku p86 (Fig. 3AGo) on the same section colocalized expression of these proteins in the region of the differentiating field of the olfactory bulb, Purkinje cell layer of the cerebellum, in a nucleus in the position of the inferior olive in the basal plate of the hindbrain (medulla), and submucosa of the nasal cavity (Fig. 3AGo) as well as in the cortex and motor neurons in the spinal cord (data not shown). Within the cerebellum and hindbrain, P450scc expression appears to be neuronal. A higher magnification of neurons within the hindbrain is shown as an insert in the panel merged.



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  		FIG. 3. Immunocytochemical analysis of Ku p86 (A) and p70 (B) subunits in the mouse nervous system. Sections of embryonic d 18.5 mouse embryos were immunostained for P450scc (green) and for either Ku p86 (red; A) or Ku p70 (red; B) on the same section. A merged image of the overlapping immunostaining is seen as yellow and is shown in the third panel. Areas in which colocalization of Ku p86 and P450scc and Ku p70 and P450scc are found include the differentiating field of the olfactory bulb (shown for Ku p86; A), the molecular layer, cortical plate and intermediate zones of the cortex (shown for Ku p70; B), the cerebellum (Purkinje cell layer, both Ku p86 and p70; arrows), the basal plate of the hindbrain (shown for both Ku p86 and p70; arrows) in the position of the inferior olive, and the nasal cavity (shown for Ku p86; A). Magnification bars, 10 µm.

 
Ku p70 and P450scc colocalized in the same distribution (Fig. 3BGo). In the cerebellum, P450scc and Ku p70 colocalized in the Purkinje cell layer. In the hindbrain (medulla), P450scc and Ku p70 colocalized in a nucleus in the position of the inferior olive. In both of these regions, P450scc expression appeared to be neuronal. P450scc and Ku p70 also colocalized in the molecular layer, cortical plate, and intermediate zones of the cortex. In the cortex, P450scc expression appeared to be glial. In the nasal epithelium, P450scc and Ku p70 were found in both epithelial cells and glandular submucosal cells (data not shown), whereas Ku p86 was found only in the glandular submucosal cells. In general, in the nervous system, Ku p86 was more widely expressed than Ku p70 or P450scc.

Transcriptional activation by Ku
We created reporter plasmids to test the ability of Ku subunits to activate transcription of the rat P450scc gene in C6 cells. These plasmids contained the sequences between -130 and +30 of the ratP450scc gene ligated to luciferase (LUC) (-130/+30rP450sccLUC, natural promoter) and a mutant plasmid of that construct containing mutations at nucleotides -120/-118, nucleotides important for Ku binding (-130/+30rP450sccLUC mutant) (15). Additional constructs were made in which the -130/-94 region was fused to a luciferase reporter containing the minimal TK32 promoter (-130/-94 TK32LUC) or with -130/-94 TK32LUC containing mutations at nucleotides -120/-118 (-130/-94 TK32LUC mutant). These four plasmids were cotransfected into C6 cells with the p70 and p86 Ku expression vectors (Fig. 4Go).



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  		FIG. 4. Transactivation of rP450scc by Ku. Rat C6 glioma cells were transfected with 1 µg of the wild-type or mutant -130/+30rP450sccLUC (natural promoter) or -130/-94rP450sccTKLUC (heterologous promoter) reporter constructs, in the absence (open bars) or presence (closed bars) of 1 µg of vectors expressing both the p70 and p86 subunits of human Ku, and luciferase activity was determined after 24 h. Mutations in both constructs were at nucleotides -120/-118. A vector lacking the -130/-94 region of the rat P450scc gene, -94/+30rP450sccLUC, was used as a control. Data, reported as relative luciferase units, are means ± SE of three experiments each done in triplicate.

 
Luciferase assays from the transfected cells showed that there was endogenous transcription of the -130/+30rP450scc-LUC reporter construct, and this transcription was greatly reduced when nucleotides -120/-118 were mutated (-130/+30rP450scc mutant). Similarly, the -130/-94TKLUC construct was transcriptionally active in C6 cells, but the construct containing mutations at -120/-118 (-130/-94 TK32LUC mutant) was also inactive. These data indicate that those nucleotides that were important for protein/DNA complex formation (15) were important for transcriptional activation in the context of the natural or heterologous (TK) promoter.

When the -130/+30rP450sccLUC and -130/-94TKLUC constructs were cotransfected the p70 and p86 Ku subunits, we did not see any change in transcription (Fig. 4Go). This could be because of the high endogenous concentration of Ku p70 and p86 subunits in C6 cells. Alternatively, it could be because Ku does not act as a transcriptional activator in those cells. Because the -130/-94 region of the rat P450scc gene was indeed transcriptionally active, we sought other candidate transcription factors, the binding of which to this region may have been masked by the abundant Ku binding.

Identification of other C6 nuclear factors that bind to the -130/-94 region of the rat P450scc gene
Because Ku binds strongly to DNA, we modified our gel-shift procedures to reduce the amount of Ku binding to our DNA by altering the buffer conditions and the order of addition of reagents, adding the radioactive probe last (24). On doing so, we identified another protein that bound to -130/-94, forming a complex that migrated more slowly than the Ku/DNA complex (Fig. 5AGo, left). The GC-rich binding site in -130/-94 was identical with an Sp1 binding site; this new band could be supershifted with an antibody to Sp1, demonstrating that the protein/DNA complex contained Sp1 (Fig. 5AGo, right, lane 1).



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  		FIG. 5. Members of the Sp transcription family bind to the -130/-94 region of the rP450scc gene. A, Effect of Sp antibodies on gel shift analysis of proteins from C6 cells binding to the -130/-94 rat P450scc oligonucleotide. Extracts from C6 cells were incubated with the rat -130/-94 probe in the presence of 200 µM MgCl2 and 200 µM ZnCl2 and radiolabeled probe was added last to reduce Ku binding (left) and then incubated in the absence or presence of 1 µl of antibody against Sp1, Sp2, Sp3, and Sp4 (right). Antihuman Sp1 antiserum supershifted the protein/DNA complex (Sp') and the Sp4 antiserum greatly reduced Sp4 binding to DNA. B, Sequence alignment of the -130/-94 region of the P450scc gene, indicating conservation of the GC-rich region. C, Gel shift analysis using probes derived from the rat or mouse P450scc sequence. Extracts from C6 cells were incubated with either the rat -130/-94 oligonucleotide or the corresponding mouse sequence. Protein/DNA complexes were competed with 100-fold molar excess of the mouse or rat oligonucleotide, as noted above the autoradiogram of the gel shift.

 
The Sp family of transcription factors consists of at least four members, Sp1–4. Gel shift analysis demonstrated that in C6 extracts, both Sp1 and Sp4 bound to the P450scc DNA because antibodies to Sp1 caused a supershift (Sp'), whereas antibodies to Sp4 inhibited protein binding (Fig. 5AGo, lanes 1 and 4). Antibodies against Sp2 and Sp3 did not ablate or cause a supershift in the protein/DNA complex, suggesting that those proteins were not found in the C6 extracts. Thus, we identified two additional proteins from C6 cells that bind to the -130/-94 region of the rat P450scc gene.

The Sp/Ku site at -130/-94 is conserved across species. Alignment of the proximal promoter sequences of the P450scc genes from rats, mice, cows, pigs, and human beings shows that each contains a sequence very similar to rat -130/-94 at a similar distance from the cap site, suggesting that this region may have conserved function across species (Fig. 5BGo). There are three nucleotide differences in this region between rats and mice, but these do not appear to mutate the Ku or Sp binding site. Gel shifts using both the rat and mouse P450scc -130/-94 oligonucleotides as probes and competitors showed that both the rat and mouse oligonucleotides formed indistinguishable protein/DNA complexes with extracts from C6 cells (Fig. 5CGo). Each of these complexes could be competed with oligonucleotides corresponding to either the rat or mouse sequence, indicating they were formed by Ku and Sp. Thus, in both rats and mice, Ku and Sp appear to participate in regulating the P450scc gene.

Sp1 stimulates transcription of the rat P450scc gene in Drosophila SL2 cells
To determine whether Sp1 could transactivate the rat P450scc gene, we used Drosophila SL2 cells, which lack Sp1, Sp2, Sp3, and Sp4 (27, 28). Cells were transfected with the -130/+30rP450sccLUC vector, the vector with mutations at -120/-118 of the P450scc gene, and vectors containing the wild-type and mutant -130/-94 rP450scc oligonucleotides, ligated to TK32LUC (-130/-94rTKLUC) (Fig. 6AGo). Our control plasmid had the -130/-94 region of the rP450scc gene deleted and cloned into the luciferase reporter vector (-94/+30rP450sccLUC). In the absence of additional Sp1-expressing plasmids, all reporter constructs yielded minimal luciferase activity. When the Sp1-expression vector was cotransfected with the -94rP450sccLUC vector, there was no activation by Sp1. When the Sp1-expression vector was cotransfected with the -130rP450sccLUC vector, there was a more than 40-fold increase in luciferase activity. This increased luciferase activity was reduced considerably when nucleotides -120/-118 of the rP450scc gene were mutated, indicating that those nucleotides were important for Sp1 transcriptional activity. Similar results were obtained when a Sp4-expression vector was used in place of the Sp1-expression vector (data not shown).



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  		FIG. 6. Transactivation of rP450scc by Sp1 and Ku. A, Effects of Sp1 on P450scc transcription. Drosophila SL2 cells were transfected with 200 ng of the wild-type or mutant -130rP450sccLUC (natural promoter) or -130/-94 rP450sccTK32LUC (heterologous promoter) reporter constructs in the absence (open bars) or presence of an expression vector expressing Sp1 (pPAC-Sp1) (100 ng) (black bars), and luciferase activity was determined after 48 h. Trinucleotide mutations in both constructs were at nucleotides -120/-118. The control plasmid deleting the -130/-94 region is called -94rP450sccLUC. Data, reported as relative luciferase units, are means ± SE of three experiments, each done in triplicate. B, Effects of both Sp1 and Ku on P450scc transcription. Drosophila SL2 cells were transfected with 200 ng of the -130rP450sccLUC or -94rP450sccLUC reporter constructs in the absence or presence of the Sp1-expression vector of vectors expressing both the p70 and p86 subunits of human Ku or both Sp1- and Ku-expression vectors, and luciferase activity was determined after 48 h. Data are means ± SE of three experiments, each done in triplicate.

 
When a heterologous TK minimal promoter replaced the natural P450scc promoter, similar results were obtained. When the Sp1 expression vector was cotransfected with the wild-type -130/-94rP450sccTKLUC, there was a comparable increase in luciferase activity. Mutation of nucleotides -120/-118 yielded a greatly reduced luciferase activity. These data indicate that Sp1 binds to the -120/-113 region of the rP450scc gene and increases its transcription, and this effect is independent of the minimal promoter.

Ku increases Sp1-stimulated transcription of the rat P450scc gene in Drosophila SL2 cells
To determine whether Ku had an effect on Sp1-stimulated transcriptional activation of the P450scc gene, we transfected Drosophila SL2 cells with the -130/+30rP450sccLUC or -94/+30rP450sccLUC vector, in the absence or presence of Ku p70/p86 and Sp1 expression vectors (Fig. 6BGo). Ku had no effect on the transcription of either the -130/+30rP450sccLUC or -94/+30rP450sccLUC vectors alone, whereas Sp1 stimulated transcription from the -130/+3rP450sccLUC reporter but not from the -94/+30rP450sccLUC reporter that lacks the Sp1 site. However, in the presence of Sp1, Ku augmented transcription from the -130/+30rP450sccLUC reporter but not from the -94/+30rP450sccLUC vector. These data suggest that Ku acts synergistically with Sp1 to increase transcription of the rat P450scc gene.

Colocalization of P450scc and Sp family members in the nervous system
To determine whether Sp factors colocalized with P450scc in the nervous system during development, we performed immunocytochemistry for P450scc and Sp1, 2, or 4 on the same section (Fig. 7Go). At embryonic d 9.5, both Sp2 and Sp4 colocalize with P450scc in the ventral neuroectoderm (neuroepithelium, arrows). Sp4 and P450scc colocalize in Rathke’s pouch, and Sp2 and P450scc also colocalize in the ventral neural tube, seen in the middle panels. P450scc and Sp2 and P450scc and Sp4 also colocalize in the branchial arches. At embryonic d 10.5 (data not shown), Sp4 and P450scc colocalize in cells that appear to be in the position of cells migrating from the neural crest, consistent with their expression in the branchial arches, an example of tissue derived from the neural crest. At embryonic d 9.5, Sp1 does not colocalize with P450scc in any region. These data indicate that different members of the Sp family are expressed in different tissues. Thus, early in development, Sp4 and Sp2, but not Sp1, may be responsible for activating P450scc transcription.



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  		FIG. 7. Immunocytochemical analysis of Sp1, 2, and 4 in the mouse nervous system. Embryonic d 9.5 mouse embryos were immunostained for P450scc (green staining) and for Sp1 (top), Sp2 (middle), or Sp4 (bottom). Colocalization of P450scc and one of the three Sps is shown as yellow in the merged images, shown in the right panels. Arrows point to regions of colocalization for P450scc and Sp2 and Sp4. These include Rathke’s pouch, the ventral neuroectoderm, ventral neural tube, neuroepithelium in the caudal region of the tail, and branchial arches. Other structures not expressing P450scc are labeled to show orientation of the embryos. Magnification bar, 1 mm.

 
Later in development, Sp1 and Sp4 colocalized with P450scc. For example, at embryonic d 18.5, Sp1, Sp4, and P450scc colocalized in the molecular layer and cortical plate layers of the neocortex, in which P450scc expression appeared to be glial; in the hippocampal neuroepithelium; the Purkinje cell layer of the cerebellum; the ganglion cell layer, inner nuclear layer, and amacrine cells of the retina; and the pituitary primordium (Fig. 8Go). In the peripheral nervous system, P450scc colocalized with Sp1 and with Sp4 in neurons in the trigeminal ganglia and dorsal root ganglia. In the whiskers, P450scc colocalized with Sp4 in the cortex and colocalized with Sp1 in the external root sheath (Fig. 9Go). Thus, different members of the Sp family may be involved in regulating P450scc gene expression at different times in development.



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  		FIG. 8. Immunocytochemical analysis of Sp1, Sp4, and P450scc expression in the embryonic d 18.5 mouse. P450scc immunocytochemistry used a FITC-labeled secondary antibody, and positive signals appear green in panels on the left. Sp1 immunocytochemistry used an Alexa-fluor 350-labeled antibody, and the positive signal appears blue in the middle panels. Sp4 immunocytochemistry used a biotinylated antibody and Cy3-conjugated Extravidin, and the positive signals appear red in the middle panels. An image of the combined immunofluorescence of all three antibodies is seen in the panels on the right, called merged. P450scc and Sp1 appear as cytoplasmic immunostaining, and Sp4 appears as nuclear immunostaining. egl, External germinal layer of the developing cerebellum; retinal cell layers; gcl, ganglion cell layer; ipl, inner plexiform layer; inl, inner nuclear layer; iloc, internal layer of optic cup; oloc, external layer of optic cup. Magnification bars for the cortex, hippocampus, cerebellum, and pituitary are the same. Bar (shown under the figure of the pituitary), 100 µm. The magnification of the retina is lower than for the other photomicrographs. Magnification bar (shown under the figure for the retina), 100 µm.

 


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  		FIG. 9. Immunocytochemical analysis of Sp1, Sp4, and P450scc expression in the peripheral nervous system at embryonic d 18.5. P450scc, Sp1, and Sp4 immunostaining is the same as described in the legend for Fig. 8Go. Magnification bars, 100 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have identified two different, transcriptionally active regions of the rat P450scc gene. The -80/-35 region is active only in steroidogenic cells such as MA-10 or Y-1 and is bound by SF-1 (15). In contrast, we have now shown that the region at -130/-94 is active in C6 glial cells and is bound by the p70 and p86 subunits of Ku as well as by members of the Sp family of transcription factors. We also found that these Ku and Sp factors are differentially and regionally expressed during development, suggesting that the developmental regulation of P450scc in the rodent nervous system involves several different transcription factors that bind to similar regions of the rat P450scc 5' flanking DNA and may work alone or together to increase P450scc gene transcription. P450scc, as well as Sp transcription factors and Ku, is expressed both in glia and neurons. Our immunocytochemical data indicate that P450scc expression early in development involves members of the Sp family because Sp family members colocalize with P450scc protein in the developing nervous system. Ku may also be involved in the transcriptional regulation of P450scc in the nervous system because both the Ku p70 and Ku p86 subunits bind to P450scc DNA and colocalize with each other and P450scc in particular regions of the nervous system. Although cotransfection of Ku p70/p86 expression vectors alone with a P450scc promoter/reporter construct did not show transactivation in C6 glioma or Drosophila SL2 cells, Ku substantially increased Sp1-stimulated transcription of the P450scc gene in the SL2 cells. Thus, because Ku is widely expressed in many cell types, it appears that its role in P450scc gene transcription may be dependent on the presence of Sp transcription factors. In other systems Ku has been shown to increase transcription by recruiting the DNA-dependent protein kinase component of Ku autoantigen to a particular region of DNA, in which it can then phosphorylate Sp1 through its DNA-dependent protein kinase activity (29).

It is unlikely that SF-1 plays a role in the regulation of P450scc in the nervous system because SF-1 is not expressed in regions of the nervous system in which P450scc is expressed. Thus, although adrenal and gonadal steroidogenesis is SF-1 dependent (reviewed in Refs. 16, 19, 21 , and 30, 31, 32, 33, 34), placental (23, 35, 36) and brain (15) steroidogenesis is SF-1 independent.

Ku was first identified in serum from patients with systemic lupus erythematosus and polymyositis-scleroderma overlap (37, 38, 39, 40, 41, 42). Ku is composed of two subunits, p70 and p86 (43), that bind to DNA in a sequence-specific manner (23, 43, 44, 45, 46, 47). Ku is the critical component of DNA-dependent protein kinase (reviewed in Refs. 29 and 48) and as such may play a role in transcriptional activation (45, 47, 49), repair of double-stranded DNA damage, and V(D)J recombination (50, 51, 52). It may also play a role as a DNA helicase or in the recruitment of the DNA-dependent protein kinase to a particular region of DNA, in which it can phosphorylate other transcription factors binding in the same region. For example, Ku participates in phosphorylation of Sp1 (29), Oct 1 (53), and the glucocorticoid receptor (46).

In addition to activating transcription by recruiting the DNA-dependent protein kinase component of Ku autoantigen to a particular region of DNA, it can then phosphorylate other transcription factors that bind to the same region, such as Sp1. Ku also directly regulates gene transcription: It decreases glucocorticoid-induced mouse mammary tumor virus transcription (45), and it increases the transcription of the human and rat P450c17 genes (23). No consensus DNA sequence for Ku binding has been established because the sequences to which Ku binds are different in the genes in which it activates transcription.

Sp1 also regulates transcription of the bovine and porcine P450scc genes (27, 34, 54, 55, 56, 57, 58, 59, 60, 61), and we now demonstrate that Sp1 regulates transcription of the rat P450scc gene. Sp1 from mouse Y-1 adrenocortical cells and primary cultures of bovine ovarian cells binds to the -111/101 region of the bovine P450scc gene, whereas SF-1 binds to the -57/-32 region of the gene (27, 34, 59). Both proteins associate with one another in vivo and in vitro (34, 59). Sp1, Sp3, and Sp4 each could stimulate activity of the bovine P450scc gene when transfected into Drosophila SL2 cells that lack endogenous Sp factors (27). Sp3, which is a transcriptional repressor in other systems, did not repress Sp1-dependent transcriptional activation. In addition, Sp1 also mediated cAMP-dependent transcriptional activation of P450scc in human NCI-H295 adrenocortical cells.

Our data show that specific members of the Sp gene family are temporally and regionally expressed with P450scc. Hence, early in development, when Sp4 and Sp2 colocalize with P450scc, they may mediate basal P450scc transcription, whereas later in development, Sp1 and Sp4 may be involved. Others have also found high levels of Sp4 expression early in central nervous system development, and Sp4 expression persists in the later stages of development (62). Mice in which the Sp4 gene has been ablated generally die within the first few days after birth, and those that survive are smaller than their wild-type littermates (62). Fertility of the female mutants appeared normal, whereas homozygous mutant males did not breed, despite having histologically intact testes containing mature sperm and intact reproductive tracks. Mutant males fail to copulate, indicating that Sp4 is required for normal male reproductive behavior. Sp4 is expressed in the vomeronasal organs, which play a role in the detection of pheromones, and in the hypothalamus (62); ablation of Sp4 in these two regions might contribute to the lack of normal male reproductive behavior. However, the authors found no gross abnormalities in either the vomeronasal organs or the hypothalamus of Sp4 mutant mice. In addition, no gross neurologic problems were reported in the Sp4-deficient mice, suggesting that another member of the Sp family may substitute for Sp4 in transcription of genes crucial for embryonic development. This scenario would be consistent with our data that demonstrate colocalization of both Sp2 and Sp4 with P450scc early in mouse development and colocalization of Sp1 and Sp4 with P450scc later in mouse development. Thus, we have identified several factors that may act synergistically to promote P450scc gene transcription in the developing nervous system.


   Footnotes
 
This work was supported by grants from the NIH (HD27970), National Science Foundation (0090995), and March of Dimes (to S.H.M.).

Current address for F.H.: Department of Medicine, University of Würzburg, Würzburg 97080, Germany.

Abbreviations: DHEA, Dehydroepiandrosterone; DHEAS, sulfated ester of dehydroepiandrosterone; DTT, dithiothreitol; FITC, fluorescein isothiocyanate; FPLC, fast performance liquid chromatography; LUC, luciferase; sPBT, PBS containing 0.2% Triton X-100; PMSF, phenylmethylsulfonyl fluoride; P450scc, P450 side-chain cleavage enzyme; PVDF, polyvinyl difluoride; SDS, sodium dodecyl sulfate; SF-1, steroidogenic factor.

Received January 27, 2003.

Accepted for publication October 14, 2003.


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