Activation of the SCPx promoter in mouse adrenocortical Y1 cells

Biochemical and Biophysical Research Communications 357 (2007) 549–553 www.elsevier.com/locate/ybbrc

Activation of the SCPx promoter in mouse adrenocortical Y1 cells Dayami Lopez a, Melissa Niesen a, Mohini Bedi a, David Hale a, Mark P. McLean

b,c,*

a Department of Molecular Medicine, University of South Florida, College of Medicine, Tampa, FL 33612, USA Department of Obstetrics and Gynecology, University of South Florida, College of Medicine, Tampa, FL 33612, USA Department of Molecular Pharmacology and Physiology, University of South Florida, College of Medicine, Tampa, FL 33612, USA b

c

Received 19 March 2007 Available online 9 April 2007

Abstract Sterol carrier protein X (SCPx) is a peroxisomal protein with both lipid transfer and thiolase activity. Treatment of mouse adrenal Y1 cells with cAMP for 24 h caused a significant induction of SCPx mRNA levels. Reporter gene studies demonstrated that treatment with cAMP and SF-1 was able to activate the SCPx promoter. Sequence analysis revealed the presence of three putative steroidogenic factor-1 (SF-1) binding motifs (designated SFB1, SFB2, and SFB3) and one CRE. Only SFB1 and SFB3 were able to bind recombinant SF-1 protein in electrophoretic mobility shift assays. The CRE was able to form a DNA/protein complex in the presence of Y1 nuclear extracts. Mutational analysis studies demonstrated that SFB3 is required for full activation of the SCPx promoter by cAMP treatment. Regulation of the SCPx gene by SF-1 and cAMP is similar to the regulatory mechanisms observed for other steroidogenic genes.  2007 Elsevier Inc. All rights reserved. Keywords: Steroidogenic factor-1; cAMP; CRE; SCPx; Adrenal cells

Sterol carrier protein X (SCPx) is a well-known member of a family of proteins characterized by having a common C-terminus identical to sterol carrier protein 2 (SCP2) [1]. SCPx can function as a lipid transfer protein due to its SCP2-like C-terminal domain [2,3]. However, the main role of SCPx comes from the activity of its N-terminal domain. SCPx has 3-oxoacyl-CoA thiolase activity involved in peroxisomal b-oxidation of branched-chain fatty acids (BCFAs) and in the formation of the CoA-esters of cholic acid and chenodeoxycholic acid from di- and trihydroxycholestanoic acid [1,4,5]. This function has been confirmed in SCP2/SCPx knockout studies [6,7]. It is currently unknown whether SCPx has a role in steroidogenesis. However, it has been determined that SCPx is abundantly expressed in adrenal tissues [8]. Intriguingly, the peroxisomes, the main site of SCPx expression, have * Corresponding author. Address: Department of Obstetrics and Gynecology, University of South Florida, College of Medicine, 12901 Bruce B Downs Blvd., MDC 37, Tampa, FL 33612, USA. Fax: +1 813 974 2480. E-mail address: [email protected] (M.P. McLean).

0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.03.194

been previously shown to be involved in steroid hormone production [9,10]. In some human peroxisomal disorders, defective removal of very long chain fatty acids causes the formation of lamellar aggregates in adrenocortical cells even at prenatal stages [11,12]. A most interesting finding was the identification of the first known patient with a natural mutation in the SCPx gene [13]. This mutation consisted of an insertion of a nucleotide at position 545 (545_546insA) leading to a frameshift and premature stop codon (I184fsX7) [13]. As a result, this patient lacked SCPx and the thiolase proteins but had normal SCP2 levels [13]. In addition to neurological problems, an accumulation of BCFAs in plasma, and excretion of abnormal bile acids, this patient presented with infertility [13]. Whether SCPx plays a role in the proper development and/or function of steroidogenic tissues requires further analysis. The aim of this study was to define the factors which regulate the expression of the SCPx gene in an adrenal cell line. The results suggest that the SCPx gene may be regulated similarly to other genes involved in steroid hormone production.

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D. Lopez et al. / Biochemical and Biophysical Research Communications 357 (2007) 549–553

Materials and methods Materials. Oligonucleotides and primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). Mouse adrenocortical Y1 and human HTB-9 bladder carcinoma cell lines were obtained from American Type Culture Collection (Rockville, MD). TRI Reagent was obtained from Molecular Research Center, Inc. (Cincinnati, OH). SYBR Green Real-time PCR supermix and Bio-Rad protein assay were obtained from Bio-Rad Labs (Hercules, CA). QuickChange Site-directed Mutagenesis Kit was purchased from Stratagene (La Jolla, CA). The T7 Sequenase DNA Sequencing kit and [a32P]dCTP (3000 Ci/mmol) were from GE Healthcare (Piscataway, NJ). Fugene 6 transfection reagent was purchased from Roche Applied Science USA (Indianapolis, IN). Murine SF-1 cDNA under the control of the cytomegalovirus (CMV) promoter (SF-1-pCMV) and the DNA-binding domain of murine SF-1 cloned as a fusion protein in the pGEX-1kT vector [glutathione-S-transferase (GST)SF-1] were generously provided by Dr. Keith L. Parker (University of Texas, Southwestern, Dallas, TX). All other chemicals were obtained from Fisher Scientific or Sigma Chemical Co. Quantitative real-time RT-PCR. Total RNA from mouse adrenal Y1 cells was isolated using TRI reagent. Two micrograms of DNA-free RNA was reverse transcribed using Promega’s reverse transcriptase system and random primers as per the manufacturer’s instructions. Real-time PCR reactions were performed using 200 ng of ssDNA, the Bio-Rad SYBR Green real-time RT-PCR kit, and the iCycler real-time PCR system. Each sample was amplified in triplicate using primers specific for the thiolase domain of the rat SCPx gene (5 0 -GCAAAGGTGGCTCTGCAGCA-3 0 and 5 0 -TTCCCCTTCCTCTTCAAG-3 0 ). The size of the SCPx fragment that is amplified using these primers is 197 bp. Primers specific for rat 18 S RNA (5 0 -GTAACCCGTTGAACCCCATT-3 0 and 5 0 -CCATCCAA TCGGTAGTAGCG-3 0 ) were used as the internal control for these studies. The parameters for PCR were: denaturation at 95 C for 3 min, followed by 39 cycles of denaturation at 95 C for 30 s, annealing at 59 C for 30 s, and extension at 72 C for 30 s. Quantitation of the results was performed using the Comparative CT method. Plasmids for transfections. To make the pLUC-1570 SCPx promoterluciferase gene construct, the 1570 bp region immediately upstream of the rat SCPx gene translation start site was obtained as described [14] and cloned into the pGL3-basic luciferase vector (Promega). The pLUC-935 SCPx construct was prepared as previously described [14]. Mutants of the pLUC-1570 SCPx construct were obtained using the QuickChange Sitedirected Mutagenesis Kit (Stratagene, La Jolla, CA) as previously described [14]. Mutagenic oligonucleotides used herein were 5 0 -CAC TTTGCAGCCTGCACCCGGCTGGACTTGTG-3 0 (for SFB-1), 5 0 GAAACTGAGGTCTCCGGGAGAAAACTGCCTG-3 0 (for SFB-3), and 5 0 -CGGCCCCGCCCCTGATTTCTGGGGCTGGGATAAG-3 0 (for CRE), and their respective complements. The nucleotides that are underlined correspond to the mutated bases. SFB refers to the SF-1 binding site. All mutations were confirmed by sequencing using the T7 Sequenase DNA sequencing kit and [35S]dATP. Reporter gene studies. Cells were transfected with the specified SCPx promoter-luciferase gene construct either in the presence or absence of SF1 expression plasmids using the Fugene 6 transfection method as previously described [14]. After transfection, cells were incubated for 48 h. Twenty-four hours prior to the end of the incubation period, some cells received 8-bromo-cAMP (8-Br-cAMP). Cell lysate preparation and luciferase assays were carried out as previously described [14]. Co-transfection of a plasmid containing the renilla luciferase gene under the control of the simian virus 40 early enhancer/promoter region was used as a control to correct for differences in transfection efficiencies. Fusion protein production. GST-SF-1 overexpressed in Escherichia coli was purified as previously described [15]. Recombinant protein samples were concentrated approximately 4- to 6-fold using Centricon-10 concentrators (Millipore, Billerica, MA). Protein concentrations were determined using the Bio-Rad protein assay. Nuclear extract preparation. Nuclear extracts from Y1 cells were prepared as previously described [14]. Concentration of protein samples and

determination of protein concentrations were performed as described above. Electrophoretic mobility shift assay (EMSA). Complementary oligonucleotides corresponding to the rat SCPx promoter regions from 1513 to 1488 (SFB-1: 5 0 -GCAGCCTGCACAAGGTGGACTTGAAT-3 0 ), from 685 to 659 (SFB-2: 5 0 -GGGAATATAAAAGGCGTAAAC CAACC-3 0 ), from 231 to 205 (SFB-3: 5 0 -AACTGAGGTCTCCTTGA GAAAACTGC-3 0 ), and from 101 to 75 (CRE: 5 0 -CCCCGCCCCTGA CGCCTGGGGCTGGGA-3 0 with GGG overhangs at the 5 0 -ends, were synthesized and annealed. Radiolabeling of annealed oligonucleotides, binding reactions and electrophoresis were carried out as previously described [15]. Where indicated, SF-1 specific antibody or unlabeled oligonucleotide (competitor) was added to the binding reactions. A known SFB from the rat HDL-R promoter [15] was used as a positive control for this assay.

Results and discussion Cyclic AMP (cAMP) activation of steroidogenic enzymes has been shown to be essential for proper maintenance of the steroid hormone biosynthetic pathway [16]. One of the mechanisms by which cAMP activates steroid hormone synthesis is by inducing expression of steroidogenic genes [16]. To specifically determine the effect of cAMP treatment on SCPx mRNA levels in the mouse adrenal Y1 cell line, quantitative real-time RT-PCR was performed. For this, RNA was prepared from Y1 cells treated with and without 1 mM 8-bromo cAMP [8-BrcAMP] for 24 h. As shown in Fig. 1A, cAMP treatment significantly (p < 0.04) increased SCPx mRNA levels by 5-fold. These results suggest that cAMP may regulate SCPx gene expression at the transcriptional level. To begin examining whether cAMP could actually alter transcription of the SCPx gene, reporter gene studies were performed in Y1 cells using the pLUC-1570 SCPx construct. The data are represented as mean relative luciferase units where the value of basal luciferase activity produced from the promoter construct in the absence of treatment is set to 1.0. As shown in Fig. 1B, adding increasing amounts of 8-Br-cAMP caused a progressive increase in luciferase activity produced from this SCPx promoter construct. Significant increases (2.5-fold; p < 0.05) in promoter activity were obtained in the presence of 1 mM 8-Br-cAMP (Fig. 1B). Maximal activation levels (3.3-fold) were obtained with 6 mM 8-Br-cAMP (Fig. 1B). Similar studies were then performed in the presence of another SCPx promoter construct, pLUC-935. Compared to pLUC-1570, the pLUC-935 SCPx promoter construct lacks the region between 1570 and 935. Removing the promoter region upstream of 935 caused a 60% reduction (p < 0.01) in basal promoter activity when compared to the pLUC-1570 (full-length) construct. To establish the actual effect of deleting this promoter region on 8-Br-cAMPdependent activation of the SCPx promoter, the data are represented as mean fold induction where the value of basal luciferase activity for each promoter deletion construct is set to 1.0. Both deletion constructs were significantly (p < 0.01) activated by 8-Br-cAMP treatment, but

D. Lopez et al. / Biochemical and Biophysical Research Communications 357 (2007) 549–553

A

(SFB3; 5 0 -CTCCTTGA-3 0 ) relative to the translation start site. SFB1 and SFB2 had 63% identity to the SFB reported for the rat (5 0 -TCAAGGCC-3 0 ) HDL-R gene [15]. SFB3 had 75% identity to the SFB reported for the rat (5 0 -CAC CTTGG-3 0 ) steroidogenic acute regulatory protein gene [18]. To determine whether SF-1 could bind the putative SFB motifs in the SCPx promoter, EMSA was performed in the presence of recombinant SF-1 protein. As shown in Fig. 2A, incubation of radiolabeled SCPx SFB1 and SFB3 probes with recombinant SF-1 resulted in the formation of two major DNA-protein complexes (lanes 5 and 11, respectively). When 250-fold molar excess of unlabeled SFB oligonucleotide was included

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the lack of the promoter region upstream of 935 resulted in a 20% reduction in overall 8-Br-cAMP activation of the SCPx gene. To examine whether the rat SCPx promoter [14] contains any motif through which cAMP could regulate this gene, sequence analysis was performed. A putative cAMP response element (CRE) was identified at position 92 (5 0 -TGACGCCT-3 0 ) relative to the translation start site. This motif had 75% identity to the CRE in the somatostatin gene (5 0 -TGACGTCA-3 0 ) [17]. For several steroidogenic genes, cAMP regulation is mediated by the orphan nuclear receptor, steroidogenic factor-1 (SF-1) [15,18,19]. Thus, the SCPx promoter was also searched for putative binding sites for this factor. Three putative SF-1 binding motifs (SFB) were identified in the SCPx promoter at positions 1504 (SFB1; 5 0 -ACAA GGTG-3 0 ), 676 (SFB2; 5 0 -AAAAGGCG-3 0 ), and 222

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Fig. 1. Effects of 8-bromo-3 0 ,5 0 -cyclic adenosine monophosphate (8-BrcAMP) treatment on SCPx expression in mouse adrenocortical Y1 Cells. (A) Total RNA was prepared from Y1 cells treated with and without 8-BrcAMP (1 mM) for 24 h and processed by quantitative real-time RT-PCR for SCPx. The results are represented as mean relative SCPx mRNA levels where the value of SCPx mRNA for the control samples (untreated cells) was set to 1.0. (B) Effects of 8-Br-cAMP on SCPx promoter activity. The pLUC-1570 SCPx promoter construct was transfected into Y1 cells as described under Materials and methods. Increasing amounts of 8-BrcAMP were added to some of the plates 24 h before lysing. The data are represented as relative luciferase units ± SEM, where the value of luciferase activity for the promoter construct in the absence of treatment is set to 1.0. These experiments were performed in triplicate and repeated three times.

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Fig. 2. Binding and activation of the rat SCPx promoter by steroidogenic factor-1 (SF-1). (A) Binding of recombinant SF-1 to the SCPx SFBs. 32Plabeled oligonucleotide probes containing the SCPx SFBs were incubated with 2.5 lg of recombinant SF-1 protein in the presence or absence of competitor (Comp; 250-fold molar excess). ‘‘+ Control’’ refers to SFB from the rat HDL-R promoter. (B) Competition analysis of the SCPx SFB1. Binding reactions were performed in the presence of 2.5 lg of recombinant SF-1 protein and increasing competing amounts (50–500·) of unlabeled SFB or SF-1 specific antibody (5–17 lg of IgG). Representative gel mobility shift assay autoradiographs are shown in (A and B). (C) SF-1 activation of the SCPx Promoter in Human Bladder Carcinoma HTB-9 Cells. Transfections in the presence of SF-1 expression plasmid and treatment with 8-Br-cAMP were performed as described in Materials and methods. The data are represented as relative luciferase units ± SEM, where the value of luciferase activity for the promoter construct transfected in the absence of SF-1 is set to 1.0. These experiments were performed in triplicate and repeated three times.

D. Lopez et al. / Biochemical and Biophysical Research Communications 357 (2007) 549–553

in the binding reactions (lanes 6 and 12, respectively), these DNA–protein complexes were completely eliminated (Fig. 2A). SF-1 protein bound to SFB1 and SFB3 with lower intensity than to the control SFB motif (Fig. 2A). No binding of recombinant SF-1 to SFB2 was observed (Fig. 2A, lanes 7–9). To further examine the specificity of SF-1 binding to the SFB1 site, competition studies were performed. As shown in Fig. 2B, the addition of increasing levels of unlabeled SFB1 oligonucleotide (10- to 250-fold; lanes 2–6) or SF-1 specific antibody (5–17 lg of IgG; lanes 7–9) gradually diminished the formation of the DNA–protein complexes. Similar results were obtained with SFB3. Binding of SF-1 to SFB1 and SFB3 was also confirmed using Y1 nuclear extracts. Interestingly, the 60% reduction in basal luciferase activity and the 20% reduction in 8-Br-cAMP-dependent activity of the SCPx promoter as a result of deleting the promoter region upstream to 935 (see above) correlated with removal of SFB1 site. To determine whether SF-1 could specifically enhance the activity of the rat SCPx promoter, reporter gene studies were performed in human bladder carcinoma HTB9 cells.

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Fig. 4. Mutational analysis of the rat SCPx promoter. (A) Schematic representation of the SCPx promoter mutated constructs employed in these experiments. (B) Transfections into Y1 cells and treatment with 8Br-cAMP (2 mM) were performed as described in Fig. 1. The data are represented as mean fold induction ± SEM, where the value of luciferase activity for each promoter mutation construct in the absence of treatment is set to 1.0. These experiments were performed in triplicate and repeated four times.

Fig. 3. Binding of Y1 nuclear extracts to the SCPx CRE. (A) Binding reactions were performed with 25 lg of Y1 nuclear protein in the presence or absence of competitor (250-fold molar excess). (B) Effects of cAMP treatment on DNA/protein complex formation. Representative mobility shift assay autoradiographs are shown in (A and B). These experiments were repeated three times.

This cell line was used because it does not express SF-1 endogenously. For this, the pLUC-1570 construct was co-transfected in the presence or absence of the SF-1pCMV plasmid. As shown in Fig. 2C, co-transfecting with SF-1 significantly increased luciferase activity produced from the promoter construct by 12-fold (p < 0.05). This activity was further enhanced by 8-Br-cAMP treatment. These results clearly demonstrate that SF-1 is directly involved in both basal and cAMP-dependent activation of the rat SCPx promoter. To examine whether the CRE motif identified in the SCPx promoter could bind any protein found in Y1 nuclear extracts, EMSA was performed. As shown in Fig. 3, the SCPx CRE motif was able to form a DNA/protein complex with the Y1 nuclear extracts. This binding was specific since adding 250-fold molar excess of unlabeled CRE eliminated the DNA–protein complex formation (Fig. 3A). However, this DNA–protein complex was not significantly affected by using nuclear extracts prepared from Y1 cells treated with 8-Br-cAMP (Fig. 3B). Incubating with an antibody specific for the CRE binding protein (CREB) had no effect on complex formation. Thus, further analysis will be required to identify the protein(s) that bind(s) to the SCPx CRE motif in Y1 cells. To confirm the role of the SCPx SFBs and the CRE motif in the cAMP-dependent activation of this gene,

D. Lopez et al. / Biochemical and Biophysical Research Communications 357 (2007) 549–553

mutational analysis was performed. The mutations analyzed were confirmed by EMSA. These mutations were then introduced into the pLUC-1570 SCPx promoter construct using site-directed mutagenesis. Fig. 4A illustrates a schematic representation of the mutated SCPx promoter constructs prepared for these experiments. No significant changes in basal promoter activity were observed for any of the mutations. As shown in Fig. 4B, mutating SFB3 and CRE independently was able to reduce cAMP-dependent activation of the SCPx promoter by 28%, while mutating SFB1 had no significant effect on cAMP-activation of this gene (Fig. 4B). Interestingly, mutating SFB1, SFB3 and the CRE reduced cAMP-dependent activation of the rat SCPx promoter by only 37% (Fig. 4B). This suggests that additional factors (cis- and trans-acting) may cooperate with the SFBs and CRE in the control of cAMP-activation of the SCPx promoter in Y1 cells. In summary, the results of the present study demonstrate for the first time that the rat SCPx gene is regulated by cAMP and SF-1 at the level of transcription in an adrenal cell line. The observed SF-1 and cAMP-dependent regulation of the SCPx gene is similar to the regulatory mechanisms observed for other genes involved in steroid hormone production. However, there is still the question of whether SCPx has a role in steroidogenesis. SCPx has both lipid transfer ability and thiolase activity, both of which could be implicated in synthesis of steroid hormones. Interestingly, SCPx has been shown to be involved in sterol absorption and synthesis of the insect moulting hormones, the ecdysteroids [20,21].

[6]

[7]

[8]

[9] [10]

[11]

[12]

[13]

[14]

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