The Transcriptional Factor Prolactin Regulatory Element-Binding ...

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Endocrinology 149(12):6103– 6112 Copyright © 2008 by The Endocrine Society doi: 10.1210/en.2008-0380

The Transcriptional Factor Prolactin Regulatory Element-Binding Protein Mediates the Gene Transcription of Adrenal Scavenger Receptor Class B Type I via 3ⴕ,5ⴕ-Cyclic Adenosine 5ⴕ-Monophosphate Koji Murao, Hitomi Imachi, Xiao Yu, Wen M. Cao, Tomie Muraoka, Hiroaki Dobashi, Naohisa Hosomi, Reiji Haba, Hisakazu Iwama, and Toshihiko Ishida Division of Endocrinology and Metabolism, Departments of Internal Medicine (K.M., H.Im., X.Y., W.M.C., T.M., H.D., T.I.), of Cardiorenal and Cerebrovascular Medicine (N.H.), and of Diagnostic Pathology (R.H.), Faculty of Medicine, and Information Technology Center (H.Iw.), Kagawa University, 1750-1, Kagawa 761-0793, Japan Prolactin regulatory element-binding (PREB) protein is a transcription factor that regulates prolactin promoter activity in the rat anterior pituitary. The PREB protein is not only expressed in the anterior pituitary but also in the adrenal gland. However, the role of PREB in the adrenal gland is not clearly understood. Scavenger receptor class B type I (SR-BI) is a receptor for high-density lipoprotein that mediates the cellular uptake of high-density lipoprotein-cholesteryl ester and is a major route for cholesterol delivery to the steroidogenic pathway in the adrenal gland. In the present study, we have examined the role of PREB in regulating SR-BI. SR-BI expression was found to be regulated by cAMP, which stimulated the expression of PREB in a dose-dependent manner.

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HOLESTEROL IS A precursor for the cellular synthesis of steroid hormones. Extracellular lipoprotein cholesterol taken up from the plasma is an important substrate source in most steroidogenic tissues (1). One source of cellular cholesterol is high-density lipoprotein (HDL), and this lipoprotein particle triggers a physiological response, leading to increased steroidogenesis (2). Consistent with this idea, lipoprotein cholesterol uptake by tumors originating from steroidogenic tissues may in fact diminish circulating cholesterol levels (3). The cellular uptake of HDL cholesterol is facilitated by a scavenger receptor class B type I (SR-BI). This protein was identified as an HDL receptor in rodents (4). The human Cluster of Differentiation 36 and lysosomal in-

First Published Online August 28, 2008 Abbreviations: Ad-LacZ, Adeno-X-lacZ adenovirus; Ad-PREB, adenovirus expressing prolactin regulatory element-binding; apo, apolipoprotein; CE, cholesteryl ester; ChIP, chromatin immunoprecipitation; CLA-1, Cluster of Differentiation 36 and lysosomal integral membrane protein II analogous-1; CRE, cAMP response element; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDL, high-density lipoprotein; hSR-BI, human scavenger receptor class B type I; NGS, normal goat serum; nt, nucleotide; PCBE, prolactin regulatory element-binding corebinding element; PKA, protein kinase A; PREB, prolactin regulatory element-binding; SF-1, steroidogenic factor-1; siRNA, small interfering RNA; SP-1, stimulatory protein 1; SR-BI, scavenger receptor class B type I; WD, tryptophon-aspartic acid. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

Conversely, overexpression of PREB using a PREB-expressing adenovirus increased the expression of the SR-BI protein in the adrenocortical cell line Y-1. In addition, PREB induced the expression of the luciferase reporter protein that was under the control of the SR-BI promoter. EMSA showed that PREB mediates its transcriptional effect by binding to the PREB-responsive cis-element of the SR-BI promoter. Finally, we used small interfering RNA to inhibit PREB expression in the Y-1 cells and demonstrated that the knockdown of PREB expression attenuated the effects of cAMP on SR-BI expression. In summary, our data showed that in the adrenal gland, PREB regulates the transcription of the SR-BI gene via cAMP. (Endocrinology 149: 6103– 6112, 2008)

tegral membrane protein II analogous-1 (CLA-1) shares 81% sequence homology with hamster SR-BI (5). Our previous reports show that human SR-BI (hSR-BI/CLA-1), like mouse SR-BI, functions as an HDL receptor (6 –14). hSR-BI/CLA-1 is also similar to the mouse homolog because it can mediate the selective uptake of the cholesterol ester, and it is expressed in the liver and steroidogenic tissues, such as the adrenal gland. These characteristics suggest that hSR-BI/ CLA-1 is functionally related to mouse SR-BI. In rodents, the highest expression of SR-BI per gram of tissue is found in the adrenal gland (4, 15). Immunostaining showed that SR-BI is expressed primarily on the surface of steroidogenic parenchymal cells, such as those in the zona fasciculata and zona reticularis of the adrenal cortex (8, 15, 16). Treatment of mice with ACTH induces adrenal steroidogenesis, enhances the selective uptake of HDL cholesterol (15), and increases SR-BI protein expression in adrenocortical cells (16). The action of ACTH on SR-BI expression is likely to be mediated by the second messenger cAMP (1). These findings support the hypothesis that in the adrenocortical tissue, SR-BI mediates the physiologically relevant selective uptake of cholesteryl esters (CEs) to provide substrates for steroid hormone synthesis. Consistent with this idea, anti-SR-BI antibodies block HDL binding and inhibit HDL-dependent steroidogenesis in cultured murine adrenocortical cells (17). Furthermore, SR-BI facilitates the entry of HDL-CEs into cultured human adrenocortical cells for steroidogenesis, thus suggesting a potential role for SR-BI in human adrenals (7). SR-BI is a

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well-characterized HDL receptor that is highly expressed in the liver and in steroidogenic tissues in rodents (4). Its human orthologue CLA-1 (hSR-BI) has also been a receptor for HDL (6), although its role in steroidogenesis is less well defined. Despite the fact that hSR-BI has not been studied as extensively as rodent SR-BI, the physiological role of hSR-BI is generally assumed to be similar to that of rodent SR-BI. The prolactin regulatory element-binding (PREB) gene encodes a 1.9-kb mRNA that translates into a transcription factor, which binds to and activates basal prolactin promoter activity (18, 19). The primary sequence of the PREB protein contains two potential trans-regulatory PQ (proline-glutamine)-rich domains and three regions of high similarity to the WD (tryptophan-aspartic acid) repeat, thus making it a member of a eukaryotic family of WD-repeat proteins. Members of this ever-expanding family of proteins are involved in multiple cellular functions that include signal transduction, RNA processing, cytoskeletal assembly, and vesicle trafficking (20). The PREB protein has similarities to a subset of proteins belonging to this family that play the role of gene regulators. Although PREB is ubiquitously expressed in human, its expression levels vary greatly among tissues, with very high levels detected in the pituitary gland, pancreas, and adrenal gland (21, 22). However, the role of PREB in the adrenal gland remains to be clarified. Furthermore, PREB participates in the protein kinase A (PKA) stimulation of prolactin promoter activity, thus suggesting that PREB plays a role in cAMP-mediated responses (18). PREB is present in the adrenal gland and is known to mediate the actions of cAMP; we speculated whether PREB participates in controlling SR-BI gene expression via cAMP. PREB protein binds directly to a conserved regulatory element in the PRL promoter via a cis-acting element, a seven nucleotide motif (ATCAGCG) corresponding to the deduced PREB core-binding element (PCBE) of the prolactin gene. Therefore, we examined the effect of PREB on the transcription of SR-BI gene expression in the adrenal gland. Our findings show that PREB binds to the promoter region of the SR-BI gene and also regulates SR-BI gene expression via cAMP. These results suggest that PREB is an important transcriptional factor that regulates the SR-BI gene in the adrenal gland. Materials and Methods Cell culture The mouse adrenocortical tumor cell line Y-1 was provided by National Institute of Health Sciences (Osaka, Japan). The cells were cultured in Ham’s F-10 medium supplemented with 15% horse serum, 2.5% fetal calf serum, 100 ␮g/ml streptomycin, and 100 U/ml penicillin in a humidified atmosphere containing 5% CO2. 8-Bromo-cAMP was purchased from Sigma-Aldrich Corp. (St. Louis, MO). Antibodies. An antibody directed against the PREB protein between amino acid residues 330 and 410 of the reported sequence (18) was generated. The corresponding cDNA fragment was amplified from rat pituitary cDNA by PCR. The amplified fragment was inserted into a pGEX-2T vector (GE Healthcare Bio-Sciences, Buckinghamshire, UK) and sequenced, and the protein was expressed in Escherichia coli. The fusion protein was isolated with glutathione-Sepharose 4B beads (GE Healthcare Bio-Sciences) and used to generate an antiserum in rabbit as described previously (6). The IgG fraction from immunized animals was purified before use in Western blot and immunohistochemistry.

Murao et al. • Role of PREB on SR-BI Expression via cAMP

Immunohistochemical localization Sections of the human adrenal gland were fixed in formalin and embedded in paraffin. After deparaffinization and rehydration, the sections were incubated in methanol containing 3% hydrogen peroxide at room temperature for 15 min to inhibit endogenous peroxidase activity. The sections were blocked for 60 min in 10% normal goat serum (NGS) in PBS and then incubated for 2 h with a guinea pig antibody against PREB (6) in 4% NGS in PBS or with preimmune antibody under identical conditions. The sections were rinsed with PBS, incubated for 30 min with a biotinylated goat antirabbit IgG (Vectastain Elite Kit; Vector Laboratories, Burlingame, CA) in 1% NGS in PBS, rinsed in PBS, and incubated with an avidin-biotinylated peroxidase complex (Vectastain Elite Kit) in PBS, as recommended by the manufacturer. Antibody binding was visualized with the diaminobenzidine reaction, and the sections were counterstained with Mayer’s hematoxylin.

Real-time PCR Template cDNA was prepared from the Y-1 cells as previously described (7). The PREB cDNA was detected by PCR using a LightCycler (Roche Diagnostics, Mannheim, Germany). The sequences of the forward and reverse hSR-BI/CLA-1 primers were 5⬘- TTGAACTTCTGGGCAAATG-3⬘ and 5⬘-TGGGGATGCCTTCAAACAC-3⬘, respectively. The sequences of the PCR primers for the rat PREB were as follows: sense primer, 5⬘-GTCATTTCCTGCCTCACT-3⬘; and antisense primer, 5⬘-GTCACATCTGTCACCACA-3⬘. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the control housekeeping gene, and was amplified and analyzed under identical conditions using previously described primers (23). The level of PREB cDNA was determined as the relative ratio of the levels of the PREB and GAPDH in the same sample.

Western blot analysis Cells were washed, scraped in PBS, and lysed as described previously (24). The proteins (15 ␮g) were separated on a 7.5% sodium dodecyl sulfate-polyacrylamide gel under reducing conditions, and transferred to polyvinylidene difluoride membranes for immunoblot assay. The membranes were incubated for 1 h at 4 C with 0.2% Tween 20 in PBS containing anti-PREB antiserum (dilution, 1:250) or anti-hSR-BI/CLA-1 antiserum (dilution, 1:2000) as described previously (6). The binding of each antibody was visualized using a chemiluminescence detection kit (enhanced chemiluminescence; Amersham Pharmacia Biotech, Buckinghamshire, UK).

Generation of adenovirus and adenovirus treatment Full-length rat PREB cDNA was inserted into the plasmid vector pShuttle as previously described (22). An adenovirus expressing PREB (Ad-PREB) was constructed using the Adeno-X Expression System kit according to the manufacturer’s instructions (Clontech Laboratories, Inc., Palo Alto, CA). As a control, Adeno-X-lacZ adenovirus (Ad-LacZ) was generated. The adenoviruses were amplified in human embryonic kidney 293 cells, and purified and concentrated to 1012 plaque-forming units per ml by cesium chloride ultracentrifugation. PREB expression in the human embryonic kidney 293 cells was induced by incubation with the Ad-PREB adenovirus for 3 h at a multiplicity of infection of 1000 plaque-forming units per cell.

Transfection of small interfering RNA (siRNA) siRNAs were designed to target the following cDNA sequences: scrambled, 5⬘-CCGTTCTGTACAGGGAGTACT-3⬘; and PREB-siRNA, 5⬘-AATGGCGTGCACTTTCTGCAG-3⬘ (22). Transfection of PREBsiRNA was performed using siPORT Amine (Ambion, Inc., Austin, TX). At 3 d after transfection, hSR-BI/CLA-1 protein expression was examined using Western blot analysis.

In vitro transcription and translation The pcDNA3.1 (⫹) vector carrying the PREB cDNA or the vector alone was transcribed in vitro using T7 RNA polymerase (Life Technologies, Inc./BRL, Tokyo, Japan) as previously described (25). The RNA



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product was translated with a rabbit reticulocyte lysate system (Promega, San Luis Obispo, CA).

notations. Alignment was performed using ReAlignerV (29) in a default setting.

EMSA

Chromatin immunoprecipitation (ChIP) assays

Nuclear extracts from the Y-1 cells were prepared according to a technique described previously (26). The synthetic DNA duplex spanning the hSR-BI/CLA-1 promoter (⫺312 to ⫺278: 5⬘-CCCCCTGCCCGTCCGATCAGCGCCCCGCCCCGTC-3⬘) (Nihon Bioservice, Asagiri, Japan) used in these studies was radiolabeled at the 5⬘-end by incubating each strand separately with [␥32P]ATP and a polynucleotide kinase before annealing. The underlined sequence indicates the PREBbinding core site. All reactions and electrophoresis were performed as previously described (26).

Y-1 cells were grown to 80 –90% confluence in 10-cm culture plates. After cross-linking for 10 min with 1% formaldehyde in serum-free medium, phosphate-glycine buffer was added to a final concentration of 0.125 m, and cells were washed twice with ice-cold PBS. The chromatin lysate was sonicated on ice to an average DNA length of 600 bp. Chromatin was precleared with blocked Sepharose A, and ChIP assays were performed with either the anti-PREB antibody or control IgG as the negative control as described by Spencer et al. (30). The final PCR step was performed to amplify the fragment spanning the nucleotides from ⫺417 to ⫺146 of the promoter sequence using the primers 5⬘-AAGAAAGAAGAGGTCGCAGG-3⬘ and 5⬘-CAGGTGGCCAGAGGCTTTAT-3⬘. After an initial 5-min denaturation at 95 C, the PCR amplification involved 32 cycles of the following steps: denaturation for 1 min at 94 C, annealing for 30 sec at 59 C, and extension for 30 sec at 72 C. Reaction products were analyzed on a 1.5% agarose-Tris-borate EDTA gel stained with ethidium bromide and visualized under UV light.

Transfection of the Y-1 cells and luciferase reporter gene assay. The reporter construct comprised the hSR-BI/CLA-1 gene sequence spanning the region from ⫺1200 to ⫹2, as determined from the published sequence (27). The segment of interest was amplified using PCR and cloned into the luciferase reporter gene (pCLA-LUC). Three constructs, pCLA2LUC, pCLA4-LUC, and pCLA6-LUC, containing promoter segments ⫺950 to ⫹2, ⫺476 to ⫹2, and ⫺209 to ⫹2, respectively, were synthesized using the parent pCLA-LUC as the template in separate PCRs. Purified reporter plasmids were transfected into the Y-1 cells (at 60% confluence) using the conventional cationic liposome transfection method (Lipofectamine; Life Technologies, Inc., Gaithersburg, MD). All assays were corrected for ␤-galactosidase activity, and the total amount of protein in each reaction was identical. For the luciferase assay, 20-␮l aliquots were used; the assay was performed according to the manufacturer’s instructions (ToyoInk, Tokyo, Japan).

Statistical analysis Statistical comparisons were made by one-way ANOVA and the Student’s t test; P ⬍ 0.05 was considered significant.

DNA analysis

Results Immunohistochemical analysis of hSR-BI/CLA-1 in the adrenal gland

To investigate whether the PCBE is conserved between human and mouse, we have aligned 510-nucleotide (nt) genomic sequences spanning 500-nt upstream of the first nucleotide site of the initiation codon and following 9 nt in the first exon of hSR-BI/CLA-1 for both the species. These genomic sequences were obtained from the latest reference genome sequences (28), NC000012.10 for human and NC000071.5 for mouse, and the particular regions were excised according to their an-

To determine the distribution of PREB expression in various cell types, we performed immunohistochemical staining of a normal adrenal gland. Figure 1A shows PREB immunostaining in the adrenal cortex. Next, we examined PREB mRNA and protein expressions in the adrenal gland and the adrenocortical cell line Y-1. Our results demonstrated the

FIG. 1. Expression of PREB in the adrenal gland. A, Immunohistochemical localization of PREB. A-I and A-II, Normal adrenal gland. PREB staining was positive in the zona fasciculata cells of the adrenal cortex. A-III and A-IV, Negative control. Representative fields were photographed at low (⫻100, panels A-I and A-III) or high (⫻1000, panel A-II and A-IV) magnification. B, RT-PCR of PREB mRNA derived from the human adrenal gland. Lane 1, Markers. Lane 2, Negative control. Lane 3, Human adrenal gland mRNA. Lane 4, Human pituitary mRNA. C, PREB protein expression. Nuclear extract from the cells was subjected to Western blot analysis. Abundance of TFIID served as a control and shown on the bottom of each lane. Lane 1, COS-7 cells as a negative control; lane 2, mouse adrenal gland; lane 3, Y-1 cells.

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FIG. 2. Effect of PREB overexpression on the expression of SR-BI. A, Western blot analysis of the effect of Ad-PREB on SR-BI protein expression. Y-1 cells were infected with Ad-PREB or Ad-LacZ for 48 h. GAPDH expression was used as the control, and the results are shown in the bottom lanes. B, A plot showing the results for each treatment group is shown in the lower panel. Results are presented as the mean ⫾ SE of three independent experiments. The asterisk denotes a significant difference (P ⬍ 0.01). LacZ, Infected with Ad-LacZ; PREB, infected with Ad-PREB.

presence of PREB mRNA of the predicted size (22) in the human adrenal gland and pituitary (Fig. 1B). As an extension of our previous finding that PREB is located in the nuclei of the cells (18), we isolated nuclear extracts from the mouse adrenal gland and Y-1 cells. The results of these studies showed the presence of a major protein band of approximately 45 kDa that matched the expected size of the PREB protein (Fig. 1C). PREB induces SR-BI protein expression

To examine whether PREB affects SR-BI expression, we measured the SR-BI protein levels in the Y-1 cells infected with the Ad-PREB. As shown in Fig. 2, the Y-1 cells infected with the Ad-PREB adenovirus showed increased SR-BI protein expression; in contrast, the expression of GAPDH was not affected. These findings suggest that in the Y-1 cells, overexpression of PREB increases the expression of the adrenal SR-BI protein. cAMP induces PREB expression in Y-1 cells

Several reports have indicated that cAMP is an important regulator of SR-BI gene expression in adrenal cells (1). Moreover, Fliss et al. (18) have reported that in pituitary cells, PREB mediates transcriptional activation of the prolactin gene via PKA. Therefore, we examined the effect of the metabolic regulator cAMP on PREB expression in the Y-1 cells. Western blots probed with a PREB-specific antiserum showed that the relative abundance of the PREB protein increased in a dose-dependent manner in response to cAMP

FIG. 3. Effects of cAMP on PREB protein and mRNA expressions in Y-1 cells. A, Nuclear extract was purified from the Y-1 cells treated with different concentrations of cAMP for 24 h. Western blot analysis was performed to examine PREB expression. Expression of TFIID was assayed as the control, and the results are shown in the bottom lanes. A plot showing the ratio of PREB level to TFIID level at each cAMP concentration is shown in the bottom panel. Results are presented as the mean ⫾ SE of three independent experiments. The asterisk denotes a significant difference (P ⬍ 0.01). B, Total RNA was extracted from the Y-1 cells treated with 1 ␮M cAMP for 24 h. Real-time PCR was performed to analyze the PREB mRNA expression. The plot shows the ratio of PREB to GAPDH mRNA. Results are represented as mean ⫾ SEM of three experiments for each treatment group. The asterisk denotes a significant difference (P ⬍ 0.01). N.S., No significant difference.

(Fig. 3A). In contrast, the basal level of the transcriptional factor TFIID (Tris-borate PBS) was not affected by cAMP. Furthermore, the relative abundance of the PREB mRNA also


increased after the treatment with cAMP in a dose-dependent manner (Fig. 3B). These results clearly suggest that cAMP stimulated PREB expression in the Y-1 cells. PREB siRNA inhibits SR-BI expression and attenuates cAMP-induced stimulation

Next, we tested whether PREB might affect the cAMPinduced SR-BI protein expression. To address this question, Y-1 cells were treated with a specific or scrambled PREB siRNA and then exposed to a fixed amount (10⫺6 m) of cAMP. Our results demonstrated that the SR-BI protein levels increased in the cells treated with the scrambled siRNA and cAMP, whereas the SR-BI protein expression was markedly reduced in the cells treated with the PREB-specific siRNA (Fig. 4). These findings suggest that the cAMP-mediated induction of SR-BI expression requires PREB. PREB expression induces hSR-BI/CLA-1 promoter activity in Y-l cells

The preceding data indicate a possible role for PREB in the activity of the SR-BI promoter, but whether PREB is directly involved in controlling the SR-BI promoter remains unclear.

FIG. 4. Effects of PREB-knockdown on SR-BI expression in Y-1 cells. PREB siRNA or scrambled (sc) siRNA was transfected into Y-1 cells with or without 1 ␮M cAMP-treatment for 24 h before cell harvesting. The upper panel shows the efficiency of siRNA on PREB expression by Western blot analysis. At 48 h after transfection, the abundance of SR-BI protein level was measured using Western blot analysis. The ratio of SR-BI to GAPDH is represented as a percentage of the control. Each data point shows the mean ⫾ SE (n ⫽ 3) of separate experiments. The asterisk denotes a significant difference (P ⬍ 0.05).


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Therefore, we cotransfected a plasmid construct (p-1200 CLA-LUC) comprising the full-length hSR-BI/CLA-1 promoter fused to the luciferase open-reading frame and a PREB-expressing plasmid into the Y-1 cells, and measured the SR-BI transcription activity. As shown in Fig. 5A, PREB expression induced a 2-fold increase in the hSR-BI promoter activity. Because the preceding data indicate the hSR-BI/ CLA-1 promoter as the site of action of PREB, we searched for a cis-acting site(s) within the DNA that may mediate the effects of cAMP by creating templates with serial deletions of the hSR-BI/CLA-1 promoter contained within pCLALUC. Constructs resulting from the deletions of 250, 725, and 992 bp from pCLA-LUC yielded the templates pCLA2-LUC, pCLA4-LUC, and pCLA6-LUC, respectively. Our results (Fig. 5B) demonstrated that PREB expression increased the activity of pCLA-LUC, pCLA2-LUC, and pCLA4-LUC but not of pCLA6-LUC. Together, these observations showed that the PREB-mediated enhancement of hSR-BI/CLA-1 promoter activity required the ⫺476 to ⫺210 fragment of the promoter. PREB is involved in cAMP-stimulated hSR-BI/CLA-1 gene expression

Next, we searched for a DNA motif within the hSR-BI/ CLA-1 promoter to which PREB could bind. Examination of the promoter sequence revealed a 7-nt motif (ATCAGCG) corresponding to the deduced PCBE of the prolactin gene (18, 22). Figure 6A shows a schematic diagram of PCBE on the human and mouse SR-BI gene. The fifth block from the right (the top row) clearly showed that the PCBE was conserved between human and mouse together with flanking two stimulatory protein 1 (SP-1) elements that were reported by Cao et al. (27). To investigate whether the binding of PREB to the seven nucleotide motif (ATCAGCG) is required for its effect on the hSR-BI/CLA-1 promoter, we used EMSA. Using in vitro transcription/translation, we synthesized the PREB protein and used EMSA to assess whether the in vitro-synthesized PREB could bind to the radiolabeled probe containing PCBE. Results (Fig. 6, B and C) showed that the synthesized PREB bound to hSR-BI/CLA-1 promoter, whereas binding to an oligonucleotide probe containing a mutation within the PCBE was greatly reduced. The formation of this complex was shifted by preincubation with antibody against PREB (Fig. 6D). Furthermore, a ChIP assay was used to determine whether PREB bound to the SR-BI promoter. Figure 6E shows the PCR amplification product after the immunoprecipitation of the cross-linked chromatin with the PREB antibody (Fig. 6E, lane 5). No PCR amplified product was found after the immunoprecipitation of the cross-linked chromatin with purified rabbit IgG (Fig. 6E, lane 4). These data support the idea that PREB binds to the SR-BI promoter, which spans the nucleotides from ⫺417 to ⫺146 in the SR-BI promoter sequence. This finding led us to create a plasmid construct, pCLA-mt-LUC, containing a mutated putative PCBE (5⬘-ATC-3⬘ to 5⬘-GCA-3⬘). Transfection studies showed that PREB failed to induce any luciferase activity in cells transfected with the pCLA-mt-LUC plasmid, but as mentioned previously, it stimulated luciferase activity in cells transfected with the wild-type pCLA-LUC plasmid (Fig. 7A).

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Together, these findings suggest that the putative PCBE in the hSR-BI/CLA-1 promoter is involved in the PREB-mediated induction of the hSR-BI/CLA-1 promoter. In addition, a mutation in PCBE inhibited the ability of cAMP to stimulate hSR-BI/CLA-1 promoter activity (Fig. 7B). The induction of PREB protein in nuclear extracts was observed at 6 h after cAMP-treatment (Fig. 7C). The PREB mRNA was maximally induced after 24 h incubation with cAMP. Treatment of the cells for 24 h with cAMP elicited a maximal induction of both protein and promoter activity of SR-BI. These results suggest that the cAMP-mediated induction of the hSR-BI/CLA-1 promoter activity requires an intact PCBE motif. Discussion

FIG. 5. Effects of PREB on SR-BI promoter activity. A, Effect of PREB on wild-type SR-BI promoter activity. Dishes (60 mm) of Y-1 cells were cotransfected with 1 ␮g ⫺1200 CLA-LUC along with the indicated amounts of the PREB expression vector. All assays were corrected for ␤-galactosidase activity, and equal total amounts of protein per reaction were used. The results are expressed as luciferase activity relative to that of control cells, which was arbitrarily set at 100. Each data point represents the mean ⫾ SE (n ⫽ 3) of separate transfections. The asterisk denotes a significant difference (P ⬍ 0.01). B, Deletion of the ⫺476 to ⫺210 fragment of the hSR-BI promoter abolished the response to PREB. Y-1 cells were transfected with 1 ␮g of several

PREB cDNA was recently isolated from a rat pituitary cDNA library, and the protein product was shown to transactivate the prolactin promoter activity (22). PREB mRNA transcripts were present not only in the pituitary, but a strong signal was also present in both the pancreas and the adrenal gland. Recently, we reported that PREB might participate in the regulation of insulin gene transcription and hormone secretion in response to glucose stimulation (26). In this report we examined the role of PREB in the regulation of SR-BI expression in the mouse adrenal cell line Y-1. We observed that overexpression of PREB increased the SR-BI/CLA-1 protein expression in the adrenocortical cell line (Fig. 1). These findings are consistent with the idea that PREB is a transcriptional factor for SR-BI/CLA-1. Several studies have identified SR-BI as the principal receptor mediating the selective uptake of HDL-CEs in the adrenal gland (4). SR-BI is a membrane receptor, which belongs to the family of class B type I scavenger receptors. In contrast to class A scavenger receptors, class B scavenger receptors display a more restricted ligand specificity. Accordingly, SR-BI binds native and modified low-density lipoprotein, anionic phospholipids, and HDL, but not polyanions (27, 31). Recent studies have indicated that HDL binding to murine SR-BI is mediated via the apolipoproteins (apos) apoA-I, apoA-II, and apoC-III (32). High levels of SR-BI mRNA and protein are expressed in steroidogenic tissues (e.g. the adrenal gland and ovary) (6, 8, 15). Immunohistochemical analysis demonstrated a high level of SR-BI expression in the zona fasciculata and zona reticularis, and a lower level of expression in the zona glomerulosa of the adrenal gland (Fig. 1). SR-BI expression is under the control of stimuli that alter sterol metabolism, such as stress and ACTH (8, 16). In mice lacking apo A1, CE accumulation in steroidogenic cells is dramatically reduced, and ACTH-stimulated corticosteroid production is inhibited, illustrating the dependence of adrenocortical cells on the HDL-CE-selective uptake pathway (34 –37). Rigotti et al. (38) reported constructs [pCLA-LUC (CLA), pCLA2-LUC (CLA2), pCLA4-LUC (CLA4), or pCLA6-LUC (CLA6)] and cotransfected with the PREB expression vector. All assays were corrected for ␤-galactosidase activity, and the total amount of protein in every reaction was identical. The results were expressed as luciferase activity relative to that of control cells, which was arbitrarily set at 100. Each data point represents the mean ⫾ SEM of four separate transfections that were performed on separate days. The asterisk denotes a significant difference (P ⬍ 0.01). N.S., No significant difference.



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FIG. 6. Binding of PREB to hSR-BI/CLA-1 promoter. A, A schematic diagram of PCBE on the human and mouse SR-BI gene. Eight highly conserved blocks were identified in the 510-nt promoter region and were shown schematically as blue color-coded stripes. Uppercase letters stand for the conserved sequences and lowercases for flanking sequences on either side. For example, the alignment of the bottom row shows that the most right-hand side conserved block included the conserved initiation codon and its neighboring sequences. B, Doublestranded labeled 34 oligonucleotides (⫺312 to ⫺278), with the sequence of either the wild-type hSR-BI/CLA-1 promoter (lanes 1 and 2) or mutant hSR-BI/CLA-1 promoter (lane 3), were incubated with the PREB protein synthesized using in vitro transcription/translation systems. Lane P, Probe alone. Lane 1, Unprogrammed reticulocyte lysate. Lane 2, Synthesized PREB along with the wild-type probe. Lane 3, Synthesized PREB along with the mutant type probe. The arrow indicates the DNA-protein complex. An identical experiment that was performed independently showed similar results. C, Competition reactions showed binding pattern of synthesized PREB to the wild-type probe with or without an excess of cold probe. Lane P, Probe alone. Lane 1, Synthesized PREB along with the wild-type probe. Lane 2, Synthesized PREB along with the wild-type probe plus 20-fold unlabeled probe. Lane 3, Synthesized PREB along with the wild-type probe plus 100-fold unlabeled probe. An identical experiment that was performed independently showed similar results. D, Supershift assay. EMSA was performed using oligonucleotides containing 34 bp as probes with an antibody against PREB. The arrow (SS) indicates the position of supershifted band. Lane P, Probe only. Lane 1, Synthesized PREB along with the probe. Lane 2, Synthesized PREB along with the probe and the antibody against PREB. Lane 3, Synthesized PREB along with the probe and control IgG. E, Recruitment of PREB to the PCBE of SR-BI promoter region. ChIP assay was used to detect the binding of PREB to the PCBE on the SR-BI promoter of the Y-1 cells. DNAs and proteins were cross-linked with formaldehyde for 10 min, and the DNA was sheared. The cross-linked protein-DNA complexes were immunoprecipitated with the anti-PREB antibody (lane 5), or with a purified rabbit IgG as a negative control (lane 4). The protein-DNA cross-links were reversed, and the purified DNAs were used for a semiquantitative PCR analysis using a primer set for amplifying the PCBE in the SR-BI promoter region (nt ⫺417 to ⫺146). PCR of the input (sample representing PCR amplification from a 1:25 dilution of total input chromatin from the ChIP experiment) is shown in lane 2. The PCR control represents the PCR amplification in the absence of DNA (lane 3). Lane M, Marker; lane Input, positive control; lane Water control, negative control; lane IgG control, negative control for PREB antibody; lane PREB ab, PREB antibody.

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FIG. 7. Site-directed mutagenesis of the PREB-binding site abrogates the response to PREB (A) or cAMP (B). The binding site was disrupted by altering three base pairs (5⬘-ATC-3⬘ to 5⬘-GCA-3⬘) in the PREB-binding site (mutant promoter) derived from the parent construct pCLA-LUC (wild-type promoter), as described in the Materials and Methods section. Cells were transfected with the wild-type or mutant promoter of the SR-BI along with cotransfection with the PREB expression vector (A) or with cAMP-treatment (B). Each data point represents the mean ⫾ SEM (n ⫽ 3) of separate transfections. The asterisk denotes a significant difference (P ⬍ 0.01). C, Time-dependent stimulation of PREB and SR-BI expression by cAMP. Y-1 cells were treated with 1 ␮M cAMP for indicated before cell harvest. Western blot and real-time PCR analysis were performed to examine PREB protein expression in nuclear (C-I) and PREB mRNA expression (C-II), respectively. SR-BI promoter activity (C-III) and protein expression (C-IV) were determined by reporter gene analysis and Western blot analysis. An identical experiment independently performed gave similar results (C-I and C-IV). A graph showing the mean ⫾ SEM of three experiments for each treatment group is shown (C-II and C-III). The asterisk denotes a significant difference (P ⬍ 0.01). N.S., No significant difference.

that mouse SR-BI provides the cholesterol substrate for steroid hormone synthesis in adrenocortical cells. These observations are supported by the results that SR-B1-deficient knockout mice have substantially reduced adrenal cholesterol stores as compared with normal controls (38). Analysis of the primary sequence of PREB reveals that it is a novel transcriptional factor, which is distinct from pituitary-specific transcription factor-1. The PREB protein has

three motifs (WD I, WD II, and WD III) with significant degrees of homology to the consensus WD repeat, suggesting that PREB is a member of the WD-repeat protein superfamily (24). The highly conserved WD repeats within PREB demonstrate sequence similarity to a subset of proteins belonging to this family, which consists of proteins that are gene regulators. Unlike other WD-repeat proteins, PREB exerts transcriptional regulation because it has stimulated gene expres-


sion by directly binding to DNA (22). Our results show that PREB binds to the SR-BI promoter and enhances the SR-BI promoter activity in Y-1 cells. The expression of SR-BI in steroidogenic cells in vivo and in vitro is regulated by trophic hormones. This regulation is mediated by the cAMP-PKA pathway (39, 40). In the SR-BI promoter, there are a number of consensus binding motifs for transcription factors (39, 40), such as CCAAT/enhancer-binding proteins, steroidogenic factor-1 (SF-1), and sterol-regulatory element-binding protein-1. It has been demonstrated that SR-BI promoter activity is increased by sterol-regulatory elementbinding protein 1␣, SF-1, estrogen, and by the phosphorylation of SP-1 (40). However, analysis of the nucleotide sequences of the human, rat, and mouse SR-BI promoter has shown that none of them has a canonical cAMP response element (CRE)binding protein responsive element (18). The absence of consensus CRE sequences in the SR-BI promoter raises the question whether ACTH-mediated stimulation occurs via the cAMP-PKA cascade (15). A recent report has indicated that stimulation of the cAMP cascade activates the SR-BI promoter via the direct phosphorylation and activation of SF-1 by PKA (41). In addition, other studies have suggested that the SF-1 level is increased after the stabilization of SF-1 protein by PKA phosphorylation (42). These reports suggest the involvement of a cAMP-PKA-mediated but CRE-binding protein-independent mechanism for the activation of the SR-BI promoter. Our results show that PREB binds to the hSR-BI/CLA-1 promoter, whereas a mutant PREB-binding site abrogates the effect of not only PREB but also cAMP. These results suggest an alternative mechanism in which PREB may be involved in the cAMP-mediated stimulation of hSR-BI/CLA-1 promoter activity. A previous report indicated that PREB could mediate the PKA-induced stimulation of prolactin promoter activity (22), thus suggesting a role for this protein in cAMP-mediated transcriptional responses. A possible model for this potential role of PREB may involve the activation of PREB via PKA-mediated phosphorylation. Figure 7C shows that the induction of PREB protein by cAMP in nuclear was observed at 6 h, before the stimulation of PREB mRNA by cAMP. Although it is not yet known whether PREB can serve as a PKA substrate either in vitro or in vivo, the predicted sequence of this protein contains a number of motifs resembling consensus PKA phosphorylation sites (33). However, the PREB-knockout in Y-1 cells showed only 60% reduction on cAMP-induced SR-BI expression (Fig. 4). One possible explanation for this observation is that other transcriptional factor(s), yet unknown, may add to net capacity of the cell to induce SR-BI expression by cAMP. In preliminary studies, adenoviral-mediated expression of PREB also stimulated the expression of steroid 11␤-hydroxylase (Murao, K., and H. Imachi, unpublished observation); this finding suggests that PREB may exert more global roles to regulate adrenal steroidogenesis. In summary, our findings show that PREB can function as a transcriptional regulator of the SR-BI/CA-1 promoter. PREB binds to the SR-BI/CLA-1 promoter, and in cells expressing PREB, it increases SR-BI/CLA-1 expression. Further investigations will help us define a possible physiological role for PREB in the adrenal gland.


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Acknowledgments Received March 19, 2008. Accepted August 19, 2008. Address all correspondence and requests for reprints to: Koji Murao, Division of Endocrinology and Metabolism, Department of Internal Medicine, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe Miki-CHO, Kita-gun, Kagawa 761-0793, Japan. E-mail: mkoji@med. kagawa-u.ac.jp. Disclosure Statement: The authors have nothing to disclose.

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