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Departments of Cell Biology and Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D.C. 20057
Address all correspondence and requests for reprints to: Dr. Vassilios Papadopoulos, Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington, D.C. 20057. E-mail: papadopv{at}georgetown.edu.
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Abstract |
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Introduction |
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PBR protein has a molecular mass of 18 kDa and is part of a multimeric complex consisting of at least another two proteins: a 32-kDa, voltage-dependent anion channel and a 30-kDa adenine nucleotide (nt) carrier (6). This multimeric complex is primarily localized at the outer mitochondrial membrane (7). However, the presence of PBR in and around the nucleus of breast cancer cells (8) and on the plasma membrane of erythrocytes (9) has been reported. The ratios of PBR, voltage-dependent anion channel, and adenine nt carrier were found to be tissue and condition specific (10).
Immunohistochemical and radioligand binding assays have detected varying amounts of PBR in all of the tissues tested. Glandular and secretory tissues, such as adrenal glands, pineal gland, salivary glands, olfactory epithelium, ependyma, and gonads are particularly rich in PBR, with renal and myocardial tissue showing intermediate levels (11, 12). In contrast, liver and brain express relatively low levels of PBR. In adrenal tissue, the medulla is virtually devoid of PBR, whereas the cortex expresses high levels (7). Similarly, the testis shows selective localization of PBR in Leydig cells, and the kidney in the distal convoluted tubules and the thick ascending lip of the loop of Henle (13).
The PBR cDNA has been cloned for a number of species, including human, rat, mouse, and cow, and in humans it encodes for a 169-amino acid protein (14, 15, 16). The high degree of homology in the coding region among different species indicates that PBR is highly conserved throughout evolution. The human and rat genes have been cloned in their entirety and were found to be composed of four exons, of which exon 1 and half of exon 4 are untranslated (17, 18). In both species the short first exon is separated from exon 2 by a large intron with several areas of repetitive sequence, which was the reason for delayed cloning of the entire gene in mouse. One transcription start site has been identified for rat and human, at position 56 and 25 nt upstream of the exon 1-intron 1 junction, respectively (17, 18). Although sequences upstream of the transcription start site have been cloned for both human and rat, important cis elements contributing to expression have not been identified yet. However, partial characterization of the rat promoter has led to the identification of one negatively and two positively acting regions for expression of the gene in Y1 adrenocortical tumor cells (19).
Besides its well established role in steroidogenesis constituting a functional component of the steroidogenic machinery that binds cholesterol and mediates its transport from the outer to the inner mitochondrial membrane (3, 20, 21, 22, 23), PBR has been found to be involved in other cellular functions, including oxidative processes, cellular proliferation, and programmed cell death. The observation of relatively high levels of PBR in several cancers, including cancers of the breast (8, 24), colon (25), ovary (26), and liver (27), suggests an additional role in tumorigenesis. Several neurological diseases, including Alzheimer’s and Huntington’s, have also been associated with changes in levels of the receptor (28, 29, 30). Finally, hypo- and hyperthyroidism have been found to affect PBR expression in heart, kidney, liver, and testis (31). In addition to these pathological conditions, PBR levels can be physiologically and pharmacologically modulated. The pituitary gland appears to regulate expression, as hypophysectomy leads to a dramatic decrease in PBR in adrenal glands, testis, and ovary (32). Similarly, adrenalectomy leads to a decrease in renal PBR levels, which can be restored after the administration of aldosterone, but not dexamethasone (33, 34). Other molecules affecting PBR expression levels include IL-1 (35); dopamine, serotonin, and norepinephrine (36); ginkgolide B (37); TNF-
(38); and several peroxisome proliferators (39).
Despite the plethora of data for the expression of PBR at the level of protein, relatively little is known about expression at the mRNA level and generally the regulation of transcription from the promoter of the gene. To better understand the regulation associated with transcription rate, detailed functional characterization of the promoter and identification of important elements for basal expression are needed. In the present study, we establish a PBR mRNA expression profile in different mouse tissues and report the presence of additional transcription initiation sites in testis and mouse Leydig tumor MA-10 cells. In addition, we report the cloning and detailed functional characterization of the mouse PBR promoter and differential utilization by two steroid hormone-producing cell lines vs. NIH-3T3 cells. Finally we investigate the importance of transcription factors Sp1 and Sp3 in transcriptional regulation of the gene.
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Materials and Methods |
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) were used to screen mouse MTC panel I (Clontech, Palo Alto, CA) and mouse tissue Rapid Scan panel (OriGene Technologies, Inc., Rockville, MD), respectively, both normalized cDNA panels according to the manufacturer’s instructions. For amplification of glyceraldehyde-3-phosphate dehydrogenase and ß-actin, the primers supplied with each panel were used. PCR products were separated by gel electrophoresis in 1.4% agarose/ethidium bromide gels and visualized under UV.
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) were used to amplify a PBR cDNA fragment extending from exon 1 to the middle of exon 3 by PCR. The fragment was purified, cloned in pCR4-TOPO (Invitrogen, Carlsbad, CA), and sequenced for verification. The plasmid was then linearized with NotI (New England Biolabs, Inc., Beverly, MA) and used as a template for in vitro transcription in the presence of biotin-14-CTP (Invitrogen) with the MaxiScript In Vitro Transcription kit (Ambion, Inc., Austin, TX) according to the manufacturer’s instructions. For the generation of ß-actin probe, plasmid pTRI-ß-actin-mouse (Ambion, Inc.) was used as a template. The ribonuclease protection assay was carried out using the Supersignal RPAIII Chemiluminescent Detection kit (Pierce Chemical Co., Rockford, IL) according to the manufacturer’s instructions. Briefly, 20 µg total RNA and 600 pg of each labeled probe were hybridized overnight and, after digestion by ribonuclease A and ribonuclease T1-protected fragments, were separated in polyacrylamide gels. These fragments were visualized using a charge-coupled device camera-based system, after transfer to nylon membranes and processing. A polyclonal antibody raised against amino acids 9–27 of the mouse PBR sequence was used for immunoblots as described previously (40) using 30 µg total cell lysate. Antibodies for Sp1 (PEP-2) and Sp3 (D-20) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Rapid amplification of 5' cDNA ends (5' RACE)
MA-10 total RNA isolated with the RNeasy kit (Qiagen, Valencia, CA) and mouse testis total RNA (Ambion, Inc.) were subjected to conventional 5' RACE and RNA ligase-mediated 5' RACE (RLM-RACE) using the Marathon cDNA amplification (Clontech) and the GeneRacer (Invitrogen) kits, respectively, according to the manufacturer’s instructions. Briefly, 1 µg total RNA was reverse transcribed with Superscript RT II (Invitrogen), and cDNA was subjected to two successive rounds of PCR with Accuprime Taq polymerase (Invitrogen) using primer R5 located in exon 4 and nested primers R1 or R2 located in exon 3 (Table 1). PCR products were separated by electrophoresis in 1.4% agarose/ethidium bromide gels and visualized under UV. Purified amplimers were cloned in pCR4-TOPO, and a total of 27 clones from conventional and 28 clones from RLM-RACE were sequenced.
Promoter cloning, luciferase reporter constructs, and expression plasmids
The mouse PBR promoter was cloned using the GenomeWalker kit (Clontech) with reverse primers GWR1 and GWR2 (Table 1) following the provided instructions. The longest PCR product obtained was purified, cloned in pCR2-Blunt II-TOPO (Invitrogen), and sequenced.
For generation of luciferase reporter constructs, the insert was freed from pCR2-Blunt II, using restriction enzymes XhoI and KpnI (New England Biolabs, Inc.) and subcloned in the corresponding restriction sites of pGL3-Basic (Promega Corp., Madison, WI) giving plasmid pGL3–2700. Plasmids pGL3–1871, -1607, -1223, -945, -805, -585, -515, -301, -214, and -123 and pGL3–15 containing 5' unidirectional deletions of the promoter were then generated from pGL3–2700 using the Erase-a-Base system (Promega Corp.) in which exonuclease III is used to digest insert DNA from a 5' protruding or blunt end restriction site.
For construction of putative alternative promoter-reporter plasmids, fragments of 1400 bp (apro1 and apro2l) and 600 bp (apro2s) were amplified by PCR using mouse genomic DNA as a template. After gel purification, the fragments were cloned in pGL3-Basic immediately upstream of the luciferase gene.
Reporter constructs pGL3-mSp1.1, -mSp1.2,3, -mSp1.4, -mAP2, and -mIk2 and pGL3-mAP1 carrying mutations in the corresponding putative transcription factor binding sites were generated with PCR and pGL3–585 as template by BioMeans, Inc. (Missouri City, TX). In all cases, the core of the putative transcription factor binding sequence was replaced with the recognition sequence of restriction enzyme NsiI.
All plasmids were fully sequenced for verification purposes.
Plasmids pPac-Sp1 and pPac-Sp3 expressing Sp1 and Sp3, respectively, under the control of the Drosophila ß-actin promoter and their backbone pPac0 were gifts from Dr. John Noti (Guthrie Research Institute, Sayre, PA).
Cell culture, transfections, and luciferase reporter assays
MA-10 cells were a gift from Dr. Mario Ascoli (University of Iowa, Ames, IA) and were maintained in DMEM/Ham’s F-12 (50:50) medium supplemented with 5% horse serum and 2.5% fetal bovine serum at 37 C and 3.7% CO2. Y1 and NIH-3T3 cells were maintained in DMEM/Ham’s F12 and DMEM, respectively, supplemented with 10% fetal bovine serum. Drosophila SL2 cells were purchased from Invitrogen, Inc. (D.Mel 2), and maintained in Drosophila serum-free medium (Invitrogen) supplemented with 18 mM L-glutamine. SL2 cells were grown in suspension at room temperature on an orbital shaker at approximately 125 rpm.
For transfection, cells were plated in six-well plates at a density of 200,000/well for MA-10 and NIH-3T3 and 400,000/well for Y1. Transfection was performed 24 h after plating, using Fugene 6 reagent (Roche, Indianapolis, IN) for MA-10 and Y1 and Polyfect reagent (Qiagen) for NIH-3T3, following the manufacturer’s instructions. All reporter plasmids were used in equimolar amounts, and pUC19 DNA was used as needed to keep the final amount of DNA constant. Plasmid pRL-TK (Promega Corp.) expressing the Renilla luciferase gene under the control of the thymidine kinase promoter was used for normalization. Twenty-four hours after transfection, the cells were washed, and total lysates were recovered using passive lysis buffer (Promega Corp). Samples were processed with the Dual-Luciferase Reporter system (Promega Corp.), and activity was measured using an automated plate reader.
Drosophila SL2 cells were plated in 24-well plates and transfected at the same time with equimolar amounts of reporter plasmids and 50 ng pPac-Sp1 or pPac-Sp3 using Fugene 6 reagent. Cotransfection with plasmid pPac0 was used to measure the background. Cells were lysed 48 h after transfection, and samples were processed and analyzed as described above. All transfection experiments were repeated at least three times in triplicate.
Preparation of nuclear extracts and EMSA
Nuclear extracts were prepared using a modified Dignam method as described previously (41). For EMSA, double-stranded 5' biotinylated oligonucleotides Sp1.2,3 and Sp1.4 (Table 1) were synthesized and incubated with 5 µg nuclear extracts from MA-10, Y1, and NIH-3T3 cells. For competition experiments, unlabeled, double-stranded oligonucleotides Sp1.2,3, Sp1.4, and mutant Sp1.2,3 and Sp1.4 (Table 1) were used. EMSA was carried out using the LightShift Chemiluminescent EMSA kit (Pierce Chemical Co.) according to the manufacturer’s instructions in the presence of EDTA and Mg2+. Complexes were separated in 6% nondenaturing polyacrylamide gels before transfer to nylon membranes, processing and visualization.
Sequence and statistical analysis
Sequence analysis was performed with Vector NTi (Informax, Invitrogen). Identification of putative transcription factor-binding sites was achieved using MatInspector version 2.2 (Genomatix, Inc., Munich, Germany). Statistical analyses were performed with PRISM version 3.0 (GraphPad, Inc., San Diego, CA).
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Results |
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Both cDNA panels showed the presence of PBR mRNA in all adult tissues examined, and in whole embryo extracts ranging from 7–17 d postcoitum (dpc; Fig. 1A). In both cDNA panels, high levels of mRNA were observed in kidney, spleen, muscle, and lung, with intermediate levels in heart and testis, and low levels in liver and brain. In addition, the Origene Technologies panel showed high levels in adrenal gland, thymus, and stomach, and intermediate levels in pancreas, uterus, and prostate (data not shown). Interestingly, whole embryo PBR mRNA levels appear to fluctuate considerably in both cDNA panels. High levels on 7 dpc are followed by low levels on 11 dpc and gradually increasing levels on 15 and 17 dpc (Fig. 1A, lanes 9–12).
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). However, steady state 18-kDa PBR protein levels are relatively high in MA-10 cells, lower in Y1 cells, and even lower in NIH-3T3 cells (Fig. 1C), suggesting differential posttranscriptional regulation of PBR. Similar to NIH-3T3, C6 rat glioma cells showed relatively high levels of mRNA, but low levels of 18-kDa PBR protein (data not shown).
Identification of the transcription initiation site(s)
Cloning of the mouse PBR cDNA by several groups revealed a slight sequence variation at the 5' end of PBR mRNA (e.g. GenBank accession nos. BC002055 and NM_009775). Here we employed conventional 5' RACE and a modified technique, termed RLM-RACE, to map the 5' end of the mouse PBR gene. Unlike conventional RACE, which favors amplification of truncated RNA, RLM-RACE amplifies only capped full-length RNA. Antisense primer R5 complementary to sequence of exons 3 and 4 and nested primers R1 and R2 complementary to sequences of exon 3 were used for two successive rounds of PCR (Fig. 2A). Conventional RACE with primers R5 and R1 or R2 using testis total RNA as template resulted in at least three amplified products, with the largest being about 350 bp in length (Fig. 2B, top), suggesting that transcription initiates at several regions of the gene. However, RLM-RACE with the same primers and template resulted in one prominent amplified product, suggesting that there is a major transcription initiation region located approximately 350 bp upstream of primer R1 (Fig. 2B, middle). RLM-RACE using MA-10 total RNA as template gave very similar results (Fig. 2B, bottom). PCR amplimers obtained from testis conventional RACE and RLM-RACE were purified, cloned in pCR4-TOPO, and sequenced. Of 27 sequenced clones from testis conventional RACE, only seven were found to initiate within exon 1, with the rest initiating within exons 2 and 3, as shown in Fig. 2C, top. However, of 26 sequenced clones from testis RLM-RACE, the overwhelming majority were found to initiate within exon 1, with one clone initiating in exon 2 and two clones initiating in exon 3 (Fig. 2C, bottom). Sequence analysis of clones initiating within exon 1 revealed that most do so at an adenine nt positioned 61 nt upstream of the translation start site (Fig. 3, arrow). These results suggest that most of the internally initiating clones derived from conventional RACE may correspond to uncapped RNA and that there is internal initiation of transcription, but possibly at a very low rate. Finally, an interesting observation was that two clones from testis conventional RACE and four clones from testis RLM-RACE, which also initiated within exon 1, were missing the 3' part of exon 2 (Fig. 2C, dashed lines), suggesting alternative splicing. These results were verified using primer extension and RPA (data not shown).
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) and used with the GenomeWalker kit. An approximately 2.7-kb promoter fragment was amplified from SspI-digested genomic DNA (data not shown), cloned in pCR2.1Blunt-TOPO, and sequenced.
Computer analysis of the cloned sequence revealed high GC content in the proximal region of the promoter and the absence of a TATA box close to the transcription start site (Fig. 3), similar to the rat PBR promoter and many other GC-rich promoters. However, a TATA box-like sequence was found at position -276. No consensus CCAAT boxes were found.
Alignment with the rat promoter revealed approximately 90% homology in the first 150 bp and approximately 80% homology in the first 1300 bp. In contrast, alignment with the human PBR promoter cloned in our laboratory revealed high homology (
70%) within the first 125 bp and only 50% homology in the first 1300 bp.
Sequence analysis led to identification of many potential binding sites for several transcription factors, including Sp1/Sp3, AP1, AP2, GATA, and members of the SOX-family (Fig. 3).
Functional characterization of the mouse PBR promoter
The functional role of the mouse PBR promoter in regulation of transcription was assessed by its ability to drive expression of the firefly luciferase gene. The approximately 2.7-kb fragment obtained from genome walking, containing most part of exon 1 (32 bp), but not the exon 1-intron 1 junction, was subcloned into pGL3-Basic, immediately upstream of the reporter gene. To measure the activity of potential cis-acting control regions and determine the minimum sequence required for maximum activity, a series of 11 reporter constructs was generated carrying progressively larger unidirectional 5' deletions of the promoter. MA-10, Y1, and NIH-3T3 cells were transiently transfected, and luciferase activity reflecting the activity of the corresponding promoter fragment was measured 24 h after transfection.
As shown in Fig. 4A, transfection of plasmid pGL3–2700 resulted in consistently high activity in all three cell lines on the order of 30–40 times the background measured using the promoterless plasmid pGL3-basic. Furthermore, similar changes in activity among the deletion constructs were observed in MA-10 and Y1 cells, with the fragment extending to -585 bp being the shortest promoter fragment that can support full activity in these cell types. Deletion of the area between -2700 and -585 bp did not have a significant effect on activity in either MA-10 or Y1 cells. However, in NIH-3T3 cells the shortest fragment supporting full activity is the one extending to -805 bp (Fig. 4A). These results suggest that sequences extending from -585 to -2700 bp and from -805 to -2700 bp may not be involved in regulation of transcription in MA-10/Y1 and NIH-3T3 cells, respectively. They also suggest that the region -585 to -805 bp is positively involved in regulation of transcription in NIH-3T3 cells. In addition, deletion of the areas between -585 and -515 bp and between -123 and -15 bp showed a significant decrease in activity in all three cell lines (Fig. 4A), indicating that these sequences may contain elements that positively regulate the expression of the gene. Fragment -15 to +36 bp showed very low activity, close to background, in all three cell lines (Fig. 4A). In COS-7 cells the shortest promoter fragment with maximum activity (20 times the background) was the one extending to -123 bp (data not shown).
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) and 600-bp (apro2s) DNA fragments located immediately upstream of the alternative transcription initiation sites were amplified by PCR from genomic DNA and cloned into pGL3-basic upstream of the firefly luciferase gene to test promoter activity. MA-10 cells were then transfected, and luciferase activity was measured 24 h later. As shown in Fig. 4B, fragments apro1 and apro2l showed no activity compared with the background, and fragment apro2s showed very low activity compared with the 2.7-kb main PBR promoter. These results in conjunction with the 5' RLM-RACE results suggest that the high rate of internal initiation of transcription is very unlikely, at least in MA-10 cells.
Putative Sp1/Sp3 binding sites in the proximal promoter are strong positive elements regulating the expression of PBR
The functional characterization of the promoter indicated that the sequence extending to -123 bp is responsible for most of the activity in both MA-10 and Y1 cells and for approximately 50% of the activity in NIH-3T3 cells. To localize and characterize strong positive elements in that area, the sequence was analyzed for identification of putative transcription factor binding sites. Four putative Sp1/Sp3, one putative AP2, and one putative Ik2 transcription factor-binding sites were found (Fig. 3). Putative Sp1/Sp3 binding sites 2 and 3 are partially overlapping and for the purpose of this study were treated as a single site, designated Sp1.2,3 (Fig. 3). Using pGL3–585 as a template, a series of plasmids was generated carrying mutations for these potential regulatory elements (Fig. 5). MA-10, Y1, and NIH-3T3 cells were then transfected with the wild-type and mutated plasmids, and luciferase activity was measured 24 h later. As shown in Fig. 5, mutation of putative regulatory element Sp1.1 did not have any effect on the activity of the promoter. However, mutation of site Sp1.2,3 resulted in a significant decrease in activity (65–70%) in all three cell lines. Mutation of site Sp1.4 led to a small, but statistically significant (P < 0.05), decrease in activity in all three cell lines. Mutation of the putative AP2 site did not have any effect, but mutation of the putative Ik2 site led to a slight decrease in activity, at least in MA-10 cells. Finally, mutation of a putative AP1-binding site located at -256 bp (Fig. 3) did not have any effect on activity, although nuclear proteins from those cell lines were able to bind that sequence in an in vitro assay (data not shown), consistent with the indication that sequences between -123 and -515 bp do not contribute significantly to the expression of the receptor in MA-10, Y1, and NIH-3T3 cells. Similar results were obtained with transfection of COS7 cells (data not shown).
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, lane 2), whereas the addition of 100-fold unlabeled Sp1.2,3 or an unrelated DNA oligonucleotide (Epstein Barr virus nuclear antigen) in the reaction indicated that complexes 1 and 2 were specific (Fig. 6A, lanes 3 and 5). Furthermore, the addition of 100-fold unlabeled mutated Sp1.2,3 indicated that proteins of specific complexes 1 and 2 do not bind the mutated sequence (compare lanes 4 and 2 in Fig. 6A). To test whether Sp1 and/or Sp3 participated in complexes 1 and 2, antibodies specific for Sp1 and Sp3 were added in the reactions. Addition of -Sp1 antibody resulted in supershifted complex 3 and a concomitant reduction in the intensity of specific complex 2 (Fig. 6A, lane 7), indicating that Sp1 is part of the latter complex. On the other hand, addition of -Sp3 antibody resulted in supershifted complex 4 and concomitant reduction in the intensity of specific complex 1, indicating that Sp3 is the main component of the latter complex. Similar results were obtained when nuclear extracts from Y1 and NIH-3T3 cells were incubated with labeled Sp1.2,3 oligonucleotide, although specific complexes 1 and 2 appeared less intense compared with MA-10 nuclear extracts (Fig. 6A, lanes 10–12). Similar results were obtained when labeled oligonucleotide Sp1.4 was incubated with nuclear extracts from MA-10, Y1, and NIH-3T3 cells (Fig. 6B).
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EMSA performed using a double-stranded oligonucleotide spanning the putative I
2 site in the proximal promoter did not result in any specific complexes when either MA-10 or Y1 nuclear extracts were used (data not shown). This suggests that although nuclear proteins may not bind this sequence, it may play a role in vivo in the general architecture of the promoter and therefore in transcription.
Sp1 and Sp3 activate transcription of the mouse PBR promoter
To further characterize the effects of Sp1 and Sp3 on the transcriptional activity of the PBR promoter, transient transfection experiments were also performed in Drosophila SL2 cells that lack proteins of the Sp family (42) and therefore constitute a good heterologous system for studying the effects of Sp1 and Sp3 on promoters. SL2 cells were cotransfected with wild-type pGL3–585, or pGL3–585 carrying mutations of the Sp1/Sp3 sites (Fig. 7) and expression plasmids for Sp1 or Sp3 (pPac-Sp1 and pPac-Sp3, respectively). Luciferase activity was measured 48 h after transfection and was plotted as fold activation over the background, which was measured after cotransfection with the empty expression vector (pPac0). Both Sp1 and Sp3, when expressed alone, were able to activate the wild-type construct (Fig. 7). Furthermore, activation by Sp1 appears to be primarily through binding to site Sp1.2,3, as the construct with that site mutated exhibited less than 50% the activity of the wild-type construct. On the other hand, all three Sp1/Sp3 sites seem to contribute to activation by Sp3 alone in varying degrees. No safe conclusion could be made regarding which protein is more potent in activating the PBR promoter, because Sp1 and Sp3 are not expressed at the same levels in SL2 cells (Fig. 8). These results confirm the importance of binding sites Sp1.2,3 and Sp1.4 in PBR promoter transcriptional activity, indicate that both Sp1 and Sp3 can activate the promoter when expressed alone, and finally confirm that binding site Sp1.2,3 plays a central role in PBR promoter activity.
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shows that MA-10 cells express Sp1 and Sp3 proteins at higher levels than Y1 cells, which contain higher levels than NIH-3T3 cells. The steroid-producing C6 rat glioma cells contain levels equal to those found in Y1 cells.
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Discussion |
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In the current study we investigated the relative tissue amounts of PBR mRNA. Our data indicate that PBR mRNA is present in all tissues tested. Steady-state mRNA levels are high in adrenal gland, kidney, spleen, skeletal muscle, and lung; are intermediate in heart and testis; and are low in liver and brain. These data agree in part with data obtained using oligonucleotide arrays to screen human tissues (http://genome-www.stanford.edu/genecards_v2.27/index.html). PBR protein levels seem to be directly proportional to mRNA levels in adrenal glands, liver, and brain, but inversely proportional in tissues such as kidney and testis. In analyzing these data one should consider the cell-specific localization of PBR within the tissues examined. For example, in testis, PBR is primarily found in Leydig cells, which constitute approximately 10% of the cells in the adult testis. Nevertheless, these observations suggest that PBR may be regulated at least in part at the transcription level in the former tissues mentioned above and posttranscriptionally in the latter tissues. Posttranscriptional regulation may also be the reason why although MA-10, Y1, and NIH-3T3 express comparable levels of PBR mRNA, NIH-3T3 cells express much less of the 18-kDa protein in comparison. Interestingly, PBR mRNA levels appear to fluctuate during embryonic life, being high at 7 and 8.5 dpc, followed by a decrease on d 9.5 and 11 dpc and then gradually increasing from 12.5 to 19 dpc. Whether levels of PBR protein follow this trend is not known.
Recently, the presence of covalent PBR polymers in cells was reported (40). These polymers were found to be induced by UV irradiation and to be able to bind PK11195 with higher capacity and decreased affinity relative to the PBR monomer. Therefore, one should be cautious when using radioligand binding assays and immunoblot analysis for the quantitation of PBR protein, as the levels of PBR polymers may vary in different cell types and may be induced from extensive exposure to normal laboratory conditions.
To better understand the regulation at the level of transcription we undertook the task of cloning and functionally characterizing the promoter of the gene. The first step in this task is identification of transcription start sites. Our data indicate that transcription initiates at a number of sites at the 5' end of the gene, making exon 1 variable in length, rather than at one site as reported previously for human (17) and rat (18). Such behavior is usually exhibited in genes that lack TATA and CCAAT boxes within the proximal promoter, as is the case with PBR. Often these genes contain the sequence TCTGA+1CT, termed initiator, spanning the transcription start site. In the case of PBR, the sequence spanning the most often used transcription start site located at position 32 nt upstream of the exon 1-intron 1 junction does not resemble the initiator.
Additionally, in the present study we report internal transcription initiation in both testis and MA-10 Leydig tumor cells, albeit at a very low rate compared with initiation at the 5' end of the gene. At least two internal initiation sites were found, one at the 3' end of exon 2 and one in the 5' end of exon 3. mRNAs initiating at these locations are capped and therefore could possibly be translated. As many potential translation start sites located downstream from the internal transcription start sites are in-frame with the translation start site in exon 2, the alternative transcripts could give rise to short proteins, partly identical to PBR. Previous studies using antibodies and photolabeled ligands for PBR have reported smaller proteins that are being recognized from these reagents (43, 44). Although transcription may be initiated from those alternative sites, sequences upstream either have no significant or very low promoter activity. Alternative initiation of transcription for PBR has been reported previously (17) in humans, but has not been studied in detail.
The expression of an alternatively spliced variant missing exon 2 in human tissues has been also reported (17). In the present study, transcripts missing 3' parts of exon 2 were found. These alternatively spliced variants, if translated, would give rise to proteins that do not resemble PBR, as they create a shift in the open reading frame. The relatively low levels of these splice variants suggest that they may represent errors of the splicing machinery.
The PBR promoter is highly conserved between mouse and rat, an expected observation because the two species are very closely related. The same comparison, however, between mouse and human indicates that the promoter is highly conserved only within the first 150 bp. The proximal promoter lacks a TATA or a CCAAT box, but is highly enriched in GC content, a phenomenon observed in many so-called housekeeping genes. A TATA box found at position -276 of the mouse promoter is conserved in rat, but not in human, and does not appear to be functional.
Functional characterization of the promoter showed similar activity in all three cell lines tested, although direct comparison between cell lines cannot be performed because of differences in transfection efficiency, background levels, and expression levels of the gene used for normalization. Sequences extending to -585 bp are needed for maximum activity in MA-10 and Y1 cells, but an extra 220 bp are needed for maximum activity in NIH-3T3 cells. These results suggest that important elements located between -585 and -805 bp may differentially regulate expression in nonsteroidogenic NIH-3T3 cells compared with steroidogenic MA-10 and Y1 cells. The first 123 bp of the promoter seem to be responsible for most of the activity observed in MA-10 and Y1 cells and for at least 50% of the activity in NIH-3T3 cells. This was somewhat expected, because that region of the promoter is the only highly conserved among human, rat, and mouse. The promoter seems to behave very similarly in MA-10 and Y1 cells, both steroidogenic mouse tumor cell lines. More detailed analysis of the proximal promoter indicated that two highly conserved Sp1/Sp3 binding sites play a crucial role in transcription from the PBR promoter. This observation was also somewhat expected, because those sequences are the only highly conserved putative transcription factor-binding sites within the first 123 bp of the promoter.
The Sp family of transcription factors consists of at least four members that share a highly conserved DNA-binding domain that consists of three zinc finger motifs (45). The members of the family show distinct expression patterns and diverse functions in different cell types. Sp1, the prototype of the family, and Sp3 are ubiquitously expressed and bind virtually identical DNA sequences, termed GC boxes, with comparable affinity. Sp1 has been found to act as a transcriptional activator for a large number of genes, including structural proteins, metabolic enzymes, cell cycle regulators, and transcription and growth factors (45, 46, 47, 48). In addition, Sp1 is essential for embryogenesis, as embryos lacking the gene show growth retardation and die in early gestation (49). Although Sp1 is an activator, Sp3 can function either as an activator or a repressor, in the latter case through competition with Sp1 for the same sites (50, 51). It appears that the function of Sp3 in a given set of conditions is determined by its ratio to Sp1 (45). In our study we demonstrated that both Sp1 and Sp3 from MA-10 cells are able to bind sequences Sp1.2,3 and Sp1.4 located in the proximal promoter of PBR specifically and efficiently. In addition, we showed that mutation of these sequences reduced the activity of luciferase reporter constructs in MA-10, Y1, and NIH-3T3 cells, with mutation of site Sp1.2,3 having a more prominent effect. Mutation of a third Sp1/Sp3 binding site (Sp1.1) did not reduce the activity of luciferase reporter constructs, although Sp1 and Sp3 proteins efficiently bind to its sequence in an in vitro assay (data not shown). Furthermore, we showed that both Sp1 and Sp3 activate the promoter of the gene when expressed alone in Drosophila SL2 cells. Activation by Sp1 appears to be exerted solely through binding to Sp1.2,3, as shown by greatly reduced activation in case the site is mutated. Activation by Sp3, on the other hand, seems to be exerted primarily through binding to Sp1.2,3, in a lower degree through binding to Sp1.4 and in a much lower degree through binding to Sp1.1. Given the possibility of redundancy, the afore-mentioned effects may be underestimated. These data are in agreement with the luciferase reporter results obtained from cell lines MA-10, Y1, and NIH-3T3 and suggest a central role for site Sp1.2,3 in regulation of basal transcription of the gene. Mutual exclusivity or cooperation for the binding of Sp1 and/or Sp3 to the proximal promoter is possible, given the close proximity of the sites.
The effect of cooperation between Sp1 and/or Sp3 binding the proximal promoter of a gene with transcription factors that bind sequences in distal parts for the regulation of transcription has been observed (48). Such cooperation may be happening in the case of the PBR promoter, as distal areas are required for full activity in all three cell lines used.
Although ubiquitously expressed, Sp1 and Sp3 levels are different in several tissues (http://genome-www.stanford.edu/genecards_v2.27/index.html). It could then be possible that basal levels of PBR expression are directly regulated by the amounts of Sp1 and/or Sp3 in different tissues. Although Sp1 appears to be expressed in comparable levels in MA-10, Y1, and NIH-3T3 cells, Sp3 is more abundant in MA-10 cells. This may be the reason why PBR mRNA levels in Y1 and NIH-3T3 cells are lower than those in MA-10 cells.
Studies to identify additional elements in the PBR promoter that have an effect in regulation of transcription of the gene and of the corresponding transcription factors mediating these effects are underway in our laboratory. These studies will help us elucidate the molecular mechanisms underlying abnormal PBR expression in several pathological conditions and potentially identify new modes of regulation.
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Abbreviations: dpc, Day postcoitum; nt, nucleotide; PBR, peripheral-type benzodiazepine receptor; RACE, rapid amplification of cDNA ends; RLM, RNA ligase mediated; RPA, ribonuclease protection assay.
Received October 6, 2003.
Accepted for publication November 13, 2003.
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), Y1 (
), and NIH-3T3 cells (
) were transfected with equimolar amounts of the different deletion constructs and 24 h later were lysed, and luciferase activity was measured. Normalization for transfection efficiency was performed using plasmid pRL-TK. Luciferase activity is shown as fold over background obtained with pGL3-Basic. Error bars represent the SEM from three independent experiments performed in triplicate. The transcription initiation site and GC boxes are shown with an arrow and open ovals, respectively. Positions -123 and -15 bp of the promoter are also shown. B, Schematic representation of position of fragments apro1, apro2l, and apro2s in the PBR gene. Fragments apro1, apro2l, and apro2s were amplified by PCR using genomic DNA as a template and were cloned in pGL3-Basic upstream of the luciferase gene. MA-10 cells were then transfected, and luciferase activity was measured 24 h later. Error bars represent the SEM from three independent experiments performed in triplicate.
