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Endocrinology Vol. 148, No. 11 5295-5304
Copyright © 2007 by The Endocrine Society

Regulation of Alternative Splicing of Liver Scavenger Receptor Class B Gene by Estrogen and the Involved Regulatory Splicing Factors

Xiaohui Zhang, Andrea N. Moor, Kathleen A. Merkler, Qiyuan Liu and Mark P. McLean

Departments of Obstetrics and Gynecology (X.Z., A.N.M., K.A.M., Q.L., M.P.L.) and Molecular Pharmacology and Physiology (M.P.L.), University of South Florida College of Medicine, Tampa, Florida 33612

Address all correspondence and requests for reprints to: Mark P. McLean, Ph.D., University of South Florida, 12901 Bruce B. Downs Boulevard, MDC037, Tampa, Florida 33612. E-mail: mmclean{at}health.usf.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The scavenger receptor class B isoforms (SR-B) type I and type II mediate the selective uptake of high-density lipoprotein cholesterol and promote reverse cholesterol transport, an important atherosclerosis protection mechanism, in the liver. Previously it was shown that the hepatic expression of SR-BI and SR-BII is regulated by estrogen. In the present study, we demonstrate that estrogen differentially regulates expression of the glycosylated and nonglycosylated forms of SR-BI and SR-BII in rat liver and hepatic cells. We report that estrogen mainly induces the down-regulation of glycosylated SR-BI and the up-regulation of nonglycosylated SR-BII. To study how estrogen regulates expression of the SR-B isoforms, we constructed a SR-B minigene containing minimal genomic sequences and were able to demonstrate that estrogen directly regulates the pre-mRNA alternative splicing of the exogenously expressed SR-B minigene in hepatic cells. Furthermore, we showed that the overexpression of splicing factors alternative splicing factor/splicing factor 2, Transformer (Tra)-2{alpha}, and Tra2β changes the splicing pattern of SR-B dramatically, whereas other splicing factors, such as heterogeneous nuclear ribonucleoprotein-G, SC-35, and arginine/serine-rich p40, had no effect. We also demonstrate that estrogen regulates Tra2β expression levels in liver cells. These studies suggest that estrogen may regulate SR-B isoform expression at both the RNA splicing and posttranslational modification levels and that, for alternative splicing regulation, estrogen may function by regulating the expression of the splicing factors alternative splicing factor/splicing factor 2, Tra2{alpha}, and especially Tra2β.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HIGH-DENSITY LIPOPROTEIN (HDL) cholesterol levels in plasma are known to be inversely correlated with the incidence of atherosclerosis and coronary heart disease (1). It is now established that scavenger receptor B (SR-B) type I is a major receptor that mediates cholesterol efflux to lipoproteins from atheromatous arteries (2) and reverse cholesterol transport from lipoprotein to liver (3, 4). Reverse cholesterol transport, in which cholesterol from peripheral tissues is packaged into HDL and transported back to the liver for eventual excretion in the bile (5, 6), is a major pathway for the clearance of excess cholesterol from the body. SR-BI is highly expressed in liver and mediates the selective uptake of cholesterol from HDL in these tissues (7). Because the liver accounts for 60–80% of total HDL-cholesterol clearance (8, 9), SR-BI is of great importance in the prevention of atherosclerosis and coronary heart disease. Consistent with this, overexpression of SR-BI in transgenic mice showed a protective effect on atherosclerosis (10), and the loss of SR-BI had negative effects on the cardiovascular system (11).

An alternatively spliced form of SR-BI, SR-BII, has been discovered in a variety of species including humans and rodents (12, 13). SR-BII differs from SR-BI within its carboxyl-terminal domain due to the mechanism of exon skipping in which a 129-neucleotide exon, exon 12, is included in SR-BI but excluded in SR-BII (reviewed in Ref. 14). This exclusion causes an open reading frame shift in the mRNA so that part of the 3'-untranslated region of SR-BI becomes the coding sequence in SR-BII. Functionally, SR-BII differs from SR-BI in subcellular localization and cellular trafficking and therefore mediates lipid uptake and influences cholesterol trafficking in a distinct manner from SR-BI (15, 16). It has been demonstrated that SR-BII promoted a 2-fold cholesterol efflux increase, compared with SR-BI (17). However, studies of SR-BII are limited and the physiological significance of SR-BII in either cholesterol homeostasis or the pathogenesis of cardiovascular disease is still unclear.

Modulation of hepatic SR-B expression is critical to understanding the mechanism underlying plasma cholesterol metabolism. One of our research interests is in SR-B expression regulation by estrogen in the liver. Estrogen, until recently, had been believed to have beneficial effects against cardiovascular disease (18). It is of great importance to have a better understanding of how estrogen regulates cholesterol metabolism through the HDL receptor SR-B. Studies have shown that estrogen treatment suppresses SR-BI expression in rat liver and cultured human hepatic cells (4, 19, 20, 21). Interestingly, Graf et al. (19) reported that estrogen treatment significantly increases SR-BII expression. However, neither the significance of this regulation nor the molecular mechanism underlying it has been clarified. One of the key steps is to examine how estrogen regulates SR-BI/SR-BII expression and how it regulates alternative splicing of the SR-B gene.

In this study, we first examined the effects of natural and synthetic estrogen on SR-BI and SR-BII expression in rat liver and human hepatic cell lines. To study the molecular mechanisms of alternative splicing of SR-B and its regulation by estrogen, we constructed a SR-B minigene and confirmed that estrogen directly regulates SR-B splicing. We also determined the splicing factors involved in this process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Twenty-five-day-old ovariectomized Sprague Dawley rats (Harlan Industries, Madison, WI) were anesthetized and a 2-cm piece of sterile SILASTIC brand laboratory tubing (Dow Corning Corp., Midland, MI) filled with approximately 100 mg of either 17β-estradiol (E2) or 17{alpha}-ethynyl estradiol (EE) powder (Steraloids, Newport, RI) was implanted sc in the dorsal region of each animal. Control animals received empty sterile SILASTIC brand implants. All protocols to treat animals were approved by the University of South Florida Animal Care Committee. Throughout the experiment, animals had free access to food and water and were housed under a 12-h dark, 12-h light cycle. After 0, 3, 7, 10, and 14 d of estrogen treatment, liver tissues were harvested, frozen in liquid nitrogen, and stored at –80 C for protein and mRNA analyses. E2 levels in blood serum were measured from all animals using a microtiter plate ELISA kit (BioCheck Inc., Foster City, CA) specific for E2 at a sensitivity of 1.0 pg/ml. Levels of the synthetic estrogen (EE) were measured using a microtiter Ecologiena ELISA kit (Abraxis, Warminister, PA) specific for EE and adapted for measuring EE levels in serum.

Immunoblotting
Rat liver extracts were prepared by homogenization of frozen tissue in separation buffer containing 20 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 0.2 M sucrose, 1 mM phenyl-methylsulfonyl fluoride, and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) as described in (22). Extracts were centrifuged at 3000 x g for 10 min at 4 C followed by a high-speed centrifugation of the supernatant at 100,000 x g for 45 min at 10 C. The resultant crude membrane pellets were lysed at 4 C in buffer containing 25 mM HEPES (pH 7.4), 0.25 mM NaCl, 0.5% (vol/vol) IGEPAL (Nonidet P-40), 0.2% (vol/vol) Triton X-100, 1 mM EGTA, 1 mM EDTA, 10 mM NaF, 0.1 mM Na3VO4, and protease inhibitor cocktail; sonicated briefly for 10 sec; and frozen at –80 C. Membrane protein concentrations were determined using the BCA assay (Pierce Biotechnology, Rockford, IL). Cultured cells were lysed using radioimmunoprecipitation assay buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 M EDTA, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, protease inhibitor cocktail]. Laemmli buffer (5 times) was added to the protein samples that were subsequently boiled for 4 min and size fractionated by electrophoresis on 10% SDS-PAGE gels (20 µg per lane for SR-BI and 30 µg for SR-BII) at 4 C. Anti-SR-BI (1:2000), anti-SR-BII (1:2000; Novus Biologicals, Littleton, CO), anticalnexin (1:2500; StressGen Biotechnologies, Victoria, British Columbia, Canada), anti-alternative splicing factor (ASF; 1:1000; Invitrogen Corp., Carlsbad, CA), anti-Transformer (Tra)-2β (1:200), or anti-β-actin (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) polyclonal antibodies were used for blotting. Films were imaged using a SensiCam camera (COOKE Corp. Ltd., Auburn Hills, MI), and densitometric analysis was performed using Labworks software (version 4.0; UVP Inc., Upland, CA). Deglycosylation of the SR-B proteins was carried out by treating 50 µg protein with 10 mU Peptide-N-Glycosidase F (PNGase F; ProZyme, Inc., San Leandro, CA) before immunoblotting to verify the N-glycosylated status of the proteins.

Construction of SRB minigene
An SR-B minigene was constructed by amplifying regions of the rat SR-B gene (GenBank accession no. NM031541) from rat genomic DNA by PCR using the following primers containing unique restriction sites (indicated in parentheses): 5'-GAATTCCTGCAGAGCGGGATGA-3' (EcoRI) and 5'-TCTAGAGGTCAAATGGCTTGCGTACCT-3' (XbaI); 5'-GCTAGCGGGCTTCCAGGCTGGTGTGT-3' (NheI) and 5'-GATATCGCAGGGGATGGAACTCTGAGA-3' (EcoRV); 5'-GATATCGGGATAAGGGGTTAGGCAGGG-3' (EcoRV) and 5'-GCGGCCGCTGGGGGCTCAGGACGTGG-3' (NotI). The restriction sites allowed cloning of the fragments into a TA cloning vector (Promega, Madison, WI) and ligating the fragments together in the correct order. Because the full-length introns 11 and 12 are 2.57 and 2.94 kb, respectively, we deleted part of the introns, reserving only the minimum necessary regions. The 190 bp at the 5' end and 683 bp at the 3' end of intron 11 and 233 bp at the 5' end and 57 bp at the 3' end of intron 12 were amplified (see Fig. 3AGo). The minigene (~1.5 kb) was inserted between the EcoRI and NotI sites of the pSG5 vector (Stratagene, La Jolla, CA), which was previously modified to contain a NotI site. The pSG5 vector, which contains the Simian virus 40 early promoter/enhancer, a rabbit β-globin intron 2, and a poly(A) signal after the multiple cloning sites, was used for cell culture transfection and in vitro analyses of alternative splicing. The construct was verified by sequencing.


Figure 3
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  		FIG. 3. SR-B minigene. A, Schematic representation. Large internal deletions in introns 11 and 12 were created leaving intact the functional junction sites required for pre-mRNA splicing at the 5' and 3' ends. B, SR-B minigene sequence. Exons 11, 12, and 13 are underlined. Deletions within introns 11 and 12 are indicated.

 
Cell culture and transfection of plasmids
The human hepatoma (HepG2), human cervical carcinoma (HeLa), and human bladder carcinoma (HTB9) cell lines obtained from American Type Culture Collection (Manassas, VA) were routinely maintained in DMEM/Ham’s F-12 (50:50) (DMEM/F12; Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, GA), L-glutamine, and 15 mM HEPES at 37 C and 5% CO2. The human breast carcinoma cell line (MCF-7) was similarly maintained in RPMI 1640 supplemented with 10% FBS, L-glutamine, and 15 mM HEPES buffer. For endogenous SR-BI/SR-BII studies and SR-B minigene transfection experiments, cells were plated at a density of approximately 5.0 x 105 cells/well in six-well plates in the above described media or for experiments using estrogen, phenol red-free media supplemented with 5% charcoal/dextran-treated FBS (HyClone, Logan, UT). Plasmids were transfected into cells using Lipofectamine 2000 reagent (Invitrogen) according to the respective manufacturer’s protocols. Expression plasmids of ASF/splicing factor 2 (SF2), arginine/serine-rich (SR) p40, heterogeneous nuclear ribonucleoprotein (hnRNP)-G, Tra2{alpha}, Tra2β, SC-35, ASF/SF2{Delta}RS, ASF/SF2{Delta}RRM, Tra2β{Delta}RS1, Tra2β{Delta}RS2, and Tra2β{Delta}RRM are gifts from Dr. I. D’Souza (University of Washington, Seattle, WA) (23).

Small interfering RNA (siRNA) transfection
Commercially synthesized AllStars negative control siRNA and Tra2β specific siRNA numbers 1 and 2 (QIAGEN Inc., Valencia, CA) were used for suppression of endogenous Tra2β. siRNA transfections were optimized in 12-well plates containing HepG2 cells at 80% confluency using DharmaFECT Reagent 1 (Dharmacon, Lafayette, CO) according to the protocol provided by the manufacturer. Forty-eight hours after transfection, cells were either lysed in radioimmunoprecipitation assay buffer for SDS-PAGE or harvested for total RNA isolation and RT-PCR analysis.

RT-PCR of SR-B minigene and endogenous SR-B cDNAs
Total RNA was prepared using TRI reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s protocol, treated with RNase-free DNase (TURBO DNA-free, Ambion Inc., Austin, TX), and purified with RNeasy columns (QIAGEN). RT PCR reactions using 1 µg of total cellular RNA from HepG2 or MCF-7 cells and the Titan One Tube RT-PCR system kit (Roche Applied Science, Indianapolis, IN) were performed. For the minigene experiments, PCR primers (IDT, Coralville, IA) consisted of oligonucleotides spanning nucleotides 1022–1042 (5'-TAATACGACTCACTATAGGGC-3') and 1068–1088 (5'-GCTGCAAATAAACAAGTTCTGC-3', which are uniquesequences in the pSG5 vector upstream and downstream of the insert. After reverse transcription for 30 min at 50 C, the following temperature profile was used for amplification: initial denaturation at 94 C for 2 min; 22 cycles at 94 C for 30 sec, 50 C for 30 sec, and 68 C for 45 sec (an extension of the elongation step by 5 sec per cycle was programed after 10 cycles), and a final extension at 68 C for 7 min. For endogenous SR-BI detection in HepG2 cells, the primers used were: 5'-TCTACCCACCCAACGAAGGCT-3' and 5'-CCTGAATGGCCTCCTTATCCT-3'. For endogenous SR-BII detection in HepG2 cells, the primers used were: 5'-TCTACCCACCCAACGAAGGCT-3' and 5'-AGAAGCGGGGTGTAGGGACTGG-3'. This second primer pair amplifies both SR-BI and SR-BII. After reverse transcription for 30 min at 50 C, the following temperature profile was used for amplification: initial denaturation at 94 C for 2 min; amplification at 94 C for 30 sec, 50 C for 30 sec, and 72 C for 60 sec (22 cycles for SR-BI, 38 cycles for SR-BII); and a final extension at 68 C for 7 min. Aliquots of the amplified products were analyzed on a 2% agarose gel and visualized using ethidium bromide staining. Densitometric analysis was performed using Labworks software.

Statistical analysis
All results are expressed as mean ± SEM. For densitometric analysis, all ratios were compared using an unpaired Student’s t test. P < 0.05 was considered significant for all analyses unless otherwise specified.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of SR-BI and SR-BII protein expression by E2 and EE in rat liver and HepG2 cells
Estrogen has been reported to regulate SR-BI and SR-BII expression in rat liver and liver cells (4, 19, 21). To confirm these results in rat liver, we treated female ovariectomized rats with natural estrogen, E2. Plasma estrogen concentrations in E2 SILASTIC brand capsule-implanted rats were 20.3 ± 6.1 pg/ml at d 0, 148.2 ± 9.4 pg/ml at d 3, 155.2 ± 43.1 pg/ml at d 7, 173.7 ± 29.3 pg/ml at d 10, and 122.6 ± 16.8 pg/ml at d 14, respectively. Immunoblotting analyses showed that over a time course of 0, 3, 7, 10, and 14 d of E2 treatment SR-BI protein levels in crude membrane preparations were reduced (Fig. 1AGo). However, the observed reduction in SR-BI was not as significant as reported in the literature (4, 19, 21). Next we treated the animals with a synthetic estrogen, EE. EE has a higher bioavailability and slower elimination in the body, so it is considered a more potent form of estrogen, as verified by estrogen serum level analysis, in which serum levels of EE were substantially higher, approximately 10- to 20-fold, compared with that of E2 (0 at d 0; 2394.4 ± 281.6 pg/ml at d 3; 1649.2 ± 174.8 pg/ml at d 7; 2740.8 ± 151.9 pg/ml at d 10, 1625.6 ± 211.7 pg/ml at d 14). A differential reduction in SR-BI levels was apparent beginning at d 3 in the EE-treated animals, whereas significant reductions were observed at a later date, d 7 and 10, for E2-treated animals (Fig. 1Go, A and B). Reduction of SR-BI expression in EE-treated animals is more significant than that observed in E2-treated animals over the entire treatment time course.


Figure 1
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  		FIG. 1. SR-BI expression regulation by E2 and EE in rat liver. A, Reduction of SR-BI protein expression by E2 and EE in rat liver. Ovariectomized rats were treated with estrogen implants or placebo for the indicated time course. Rat liver membrane proteins were isolated and subjected to Western blot analyses. Primary antibodies to SR-BI detected a major band at approximately 82 kDa. The endoplasmic reticulum-resident protein calnexin was used as a control for equal protein loading and is shown representatively. B, Densitometric analyses were performed on A and the ratio of SR-BI to calnexin were plotted. Data represent mean + SEM (n = 4 for each day). *, Significantly lower than the same day empty implant control (P < 0.05); **, significantly lower than the same day empty implant control (P < 0.01).

 
It has been shown that SR-BI proteins are N-glycosylated (24, 25). We observed in rat liver, as well as in HepG2 cells, that there exist glycosylated and nonglycosylated forms of both SR-BI and SR-BII, that was verified by Peptide-N-Glycosidase F digestion (Fig. 2AGo). Nonglycosylated SR-BI was reduced with EE treatment, but the changes were not completely parallel to that observed for glycosylated SR-BI (Fig. 2Go, B and C). To our surprise, unlike glycosylated SR-BII, nonglycosylated SR-BII protein levels were elevated during the estrogen treatment time course beginning at d 7 (Fig. 2Go, B and C). These data suggest that posttranslational modification of SR-BI/SR-BII proteins may play a role in their regulation by estrogen. Despite this, the significant down-regulation of total protein levels of SR-BI and perceivable up-regulation of total protein levels of SR-BII by estrogen, especially the potent form of EE, can be concluded from our data.


Figure 2
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  		FIG. 2. Glycosylated and nonglycosylated SR-BI and SR-BII expression regulation by EE in rat liver and HepG2 cells. A, Western blots show that both glycosylated (g-SR-BI and g-SR-BII) and nonglycosylated (ng-SR-BI and ng-SR-BII) forms of SR-BI and SR-BII proteins are present in HepG2 cell protein preparations. Peptide-N-Glycosidase F (PNGase F) was used to treat the proteins before immunoblotting. B, Glycosylated and nonglycosylated SR-BI and SR-BII protein expression changes with EE treatment. Calnexin was used as a control for equal protein loading and is shown representatively. C, Densitometric analyses were performed and the ratios of SR-B proteins to calnexin are plotted. Data represent mean + SEM (n = 3 for each day). # ({diamond}), * ({square}), {ddagger} ({Delta}), significantly lower than d 0 within the same group (#, P < 0.01; * and {ddagger}, P < 0.05). D, Western blots show glycosylated (g-SR-BI and g-SR-BII) and nonglycosylated (ng-SR-BI and ng-SR-BII) SR-B protein expression after treatment with EE. HepG2 cells were transiently transfected either with plem-ER{alpha} or the empty vector plem and treated for 24 h with increasing doses of EE after 24 h starvation in DMEM/F12 medium supplemented with 5% charcoal/dextran-treated FBS after transfection. β-Actin was used as a control for equal protein loading. E, Densitometric analyses were performed on glycosylated SR-BI and nonglycosylated SR-BII. Ratios of g-SR-BI to β-actin and ng-SR-BII to β-actin are used in the plot. Values of EE-treated samples are relative to those of the untreated samples, which are set to 1. Mean + SEM from three independent experiments are presented. *, Differences are significant, compared with vehicle control (P < 0.05). Densitometric results on nonglycosylated SR-BI and glycosylated SR-BII showed no significant changes (P > 0.05) (not shown).

 
Because our data from rat liver are not in line with a previous report in HepG2 cells (19), we next examined how estrogen regulates SR-BI and SR-BII expression in the HepG2 cells. Initially, we failed to show significant changes of SR-BI and SR-BII protein expression with EE treatment in HepG2 cells, whereas an increased SR-BI expression was always inducible by estrogen in MCF-7 cells (data not shown). We proposed that this might be due to reduced responsiveness in our HepG2 cells to estrogen in culture. Thus, plem-ER{alpha} was introduced into the HepG2 cells and ER{alpha} expression was increased about 4-fold (data not shown). In cells overexpressing ER{alpha}, a clear dose-dependent decrease of endogenous glycosylated SR-BI induced by EE was observed (Fig. 2Go, D and E), but no obvious decrease in the nonglycosylated form was detected, possibly due to marginal changes in nonglycosylated SR-BI that is too subtle to detect in cytosolic protein preparations. A dose-dependent increase in endogenous SR-BII induced by EE was observed in the nonglycosylated form but not the glycosylated form (Fig. 2Go, D and E). These results closely conform to the data from rat liver and emphasize the importance of ER{alpha} levels in mediating the estrogen regulation of SR-B protein expression in hepatic cells.

SR-B minigene construction and its transcription regulation by EE
To further investigate the mechanism of the differential regulation of SR-BI and SR-BII by estrogen, we constructed a SR-B minigene in the pSG5 vector as described in Materials and Methods. SR-BII differs from SR-BI due to exon 12 skipping (14). To analyze the functional exon-skipping region between exons 11 and 13 of the SR-B pre-mRNA, the minigene must contain the minimum necessary genomic regions adjacent to exon 12. The final construct contains a 1.5-kb SR-B minigene insert, which included five elements: exon 11 (149 bp), modified intron 11 (190 bp at 5'end and 683 bp at 3' end, 1.7 kb deleted in between), exon 12 (129 bp), modified intron 12 (233 bp at 5' end and 57 bp at 3' end, 2.6 kb deleted in between), and exon 13 (120 bp) (Fig. 3Go, A and B). The minigene was transfected into four different human cell lines: HepG2, HeLa, MCF-7, and HTB9 cells. Sequences in the pSG5 vector upstream and downstream of the minigene insert were used as primers to amplify the cDNA transcribed from the pSG5-minigene mRNA by RT-PCR. This excluded the amplification of the endogenous SR-B gene. Expression of SR-BI and SR-BII from the minigene in HepG2 cells resembles that of endogenous SR-B (12). In addition, the different ratios of SR-BI and SR-BII observed in the four cell lines indicate that the SR-B splicing machinery is cell type specific (Fig. 4AGo). Treatment of HepG2 cells with different doses of EE after transient transfection with the minigene produced a dose-dependent increase in the SR-BII to SR-BI expression ratio of the minigene-derived splice forms (Fig. 4BGo, upper panel, and Fig. 4DGo, left panel). The changes in SR-B minigene splice form levels were similar to the changes seen in endogenous SR-B mRNA levels in HepG2 cells treated with EE (Fig. 4Go, C and D). Furthermore, a change in the expression ratio of minigene derived SR-BII/SR-BI splice forms was also observed in MCF-7 cells (Fig. 4BGo, lower panel), with a pattern consistent with the increased protein level changes with estrogen treatment observed in MCF-7 cells. These results verify that the SR-B pre-mRNA splicing is directly regulated by estrogen. The results also validate the ability of the minigene constructs to respond to estrogen and that it can be used as a target for further exploration of the estrogen regulation of SR-B isoform expression.


Figure 4
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  		FIG. 4. SR-B minigene expression profile. A, SR-B minigene expression and splicing in multiple cell lines. SR-B minigene plasmid (M) or empty pSG5 vector (V) was transiently transfected into the indicated human cell lines. Cells were harvested after 48 h and total RNA was subjected to RT-PCR. Amplified SR-BI and SR-BII splice forms and transcript from the vector are depicted. β-Globin was used as an internal control and was amplified from a separate region within the pSG5 vector. B, Regulation of SR-B minigene expression by EE. HepG2 cells (upper panel) and MCF-7 (lower panel) cells were transiently cotransfected with SR-B minigene and ER{alpha}, and then treated with EE at the indicated dosages for 9 h after a 48-h incubation in DMEM/F12 supplemented with 5.0% charcoal/dextran-treated FBS. SR-B minigene expression was examined by RT-PCR. C, Regulation of endogenous SR-B mRNA expression by EE in HepG2 cells. HepG2 cells were transiently transfected with ER{alpha} and treated with indicated doses of EE for 24 h after a 48-h incubation in DMEM/F12 supplemented with 5.0% charcoal/dextran-treated FBS. Total RNA was isolated and subjected to RT-PCR. S16 mRNA levels were used as an internal control. Representative results from three independent experiments are presented. *, SR-BII to SR-BI ratio is significantly different, compared with vehicle control (P < 0.01). #, SR-BII to SR-BI ratio is significantly different, compared with vehicle control (P < 0.05).

 
Splicing factors involved in SR-B gene alternative splicing
Mammalian gene splicing regulation involves both cis- and trans-factors, which are composed of auxiliary element sequences in the pre-mRNA, known as splicing enhancers and silencers (26), and cellular splicing factors, which include the hnRNP, arginine/serine-rich (SR) proteins such as ASF/SF2 and SRp40, and SR-related proteins such as Tra2{alpha} and Tra2β (27). In this study, we examined the splicing factors that may possibly be involved in SR-B alternative splicing. We cotransfected an ASF/SF2, SRp40, hnRNP-G, Tra2{alpha}, Tra2β, or SC-35 expression plasmid together with the SR-B minigene into HepG2 cells. Overexpression of Tra2{alpha} and, especially, Tra2β caused a shift from the SR-BI to SR-BII splice form, increasing the ratio of SR-BII to SR-BI from 1.4 to 8.9 with Tra2β and 2.6 with Tra2{alpha}, whereas overexpression of ASF/SF2 caused a shift from SR-BII to SR-BI, reducing the ratio of SR-BII to SR-BI from 1.4 to 0.6 (Fig. 5AGo). Overexpression of SRp40, hnRNP-G, and SC-35 did not appear to have dramatic effects on SR-B splicing. We also used specific siRNA to deplete the endogenous Tra2β expression to establish the in vivo requirement for Tra2β in SR-B splicing. The depletion of Tra2β caused a reverse shift from the SR-BII to SR-BI splice form, decreasing the ratio of SR-BII to SR-BI from 1.4 to 0.4 (Tra2β siRNA number 1) or 0.8 (Tra2β siRNA number 2). The decrease in the ratio conformed to the different down-regulation levels of the protein due to the different knockdown efficiencies of the siRNA sequences (Fig. 5Go, B and C). These results indicate that ASF/SF2, Tra2{alpha}, and Tra2β, especially Tra2β, may play crucial roles in the SR-B alternative splicing machinery.


Figure 5
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  		FIG. 5. Splicing factors involved in SR-B alternative splicing. Panel A, Effects of splicing factor overexpression on SR-B splicing. SR-B minigene was cotransfected with splicing factor expression plasmids as indicated. Cells were harvested 48 h after transfection and RNA was subjected to RT-PCR analysis. Ratios of the SR-BII to SR-BI splice forms are illustrated below the gel image. Mean + SEM from three independent experiments are graphically illustrated below the gel image. Panel B, Knockdown of Tra2β expression in HepG2 cells. Protein from cells transfected with two different Tra2β-specific siRNA (Si-1 and Si-2), or negative control siRNA (C) was subjected to immunoblotting. β-Actin was used as a control for equal protein loading. Panel C, Knockdown effects of Tra2β on SR-B splicing. SR-B minigene was cotransfected with negative control siRNA (C) or Tra2β siRNA number 1 (Si-1) or number 2 (Si-2). Cells were harvested 48 h after transfection, and RNA was subjected to RT-PCR analysis. Ratios of the SR-BII to SR-BI splice forms are graphically illustrated below the gel image. Mean + SEM from three independent experiments are presented.

 
Because ASF/SF2 and Tra2β contain N-terminal RNA recognition motif (RRM) domains and C-terminal arginine-serine rich (RS) domains and these domains are essential for binding with specific RNA sequences and protein-protein interactions respectively (26, 28), we next determined which domains in the proteins contribute significantly to the SR-B splicing regulation. Individual RRM or RS domain deletion constructs for ASF/SF2 or Tra2β (23) were used in coexpression assays. For ASF/SF2, the RRM domain deletion but not the RS domain deletion reversed the SR-BII to SR-BI splice form ratio, compared with wild-type ASF/SF2 (Fig. 6Go). For Tra2β, deletion of any of the three domains eliminated the effect of Tra2β on SR-B alternative splicing (Fig. 6Go), indicating that the domains RS1, RS2, and RRM are all essential in the Tra2β effect on SR-B alternative splicing. Thus, we concluded that in SR-B alternative splicing, the ASF/SF2 protein function is mainly dependent on the RRM domain, whereas Tra2β function is dependent on both the RS and RRM domains, suggesting that ASF/SF2 mainly exerts its effect through protein and pre-mRNA interactions, whereas Tra2β needs to interact with both the pre-mRNA and other proteins to regulate SR-B alternative splicing.


Figure 6
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  		FIG. 6. Determinant domains in ASF/SF2 and Tra2β and their function in SR-B splicing. SR-B minigene was cotransfected with ASF/SF2 and Tra2β RS or RRM domain deletion constructs. Cells were harvested 48 h after transfection and RNA was subjected to RT-PCR analysis. Ratios of SR-BII to SR-BI splice forms are plotted below the gel image. Mean + SEM from three independent experiments are presented.

 
Regulation of Tra2β expression by EE
EE regulates SR-B pre-mRNA splicing and, at the same time, ASF/SF2 and Tra2β are essential splicing factors needed in SR-B splicing. To determine whether estrogen affects SR-B splicing through the regulation of splicing factor expression, we further examined the expression of ASF/SF2 and Tra2β after treatment by EE in rat liver and HepG2 cells. In rat liver, the Tra2β expression was increased on d 3 and 7 after EE treatment, compared with the control animals treated with placebo (Fig. 7Go, A and B). EE treatment for 24 h in HepG2 cells also induced an increase in Tra2β expression (Fig. 7Go, C and D). EE did not regulate the expression of ASF/SF2 protein levels in rat liver tissues and HepG2 cells (data not shown). Together with the results from the Tra2β overexpression assays, we can conclude that estrogen affects SR-B splicing at least partly through the regulation of Tra2β expression.


Figure 7
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  		FIG. 7. Estrogen treatment results in increased Tra2β expression in rat liver tissues and HepG2 cells. A and B, Tra2β expression regulation by EE in rat liver tissues. Ovariectomized rats were injected with estrogen or placebo for the indicated time course. Rat liver tissue lysates were prepared and subjected to Western blot analyses. Densitometric analyses of the bands were performed and ratios of Tra2β to β-actin are presented. Mean + SEM (n = 2 for d 0, n = 3 for the rest) are presented. *, Significantly higher, compared with d 3 group treated with placebo (P < 0.01); **, significantly higher, compared with d 7 group treated with placebo (P < 0.05). C and D, Tra2β expression regulation by EE in hepatic HepG2 cells. After starvation for 48 h in DMEM/F12 medium supplemented with 5.0% charcoal/dextran-treated FBS, HepG2 cells were treated with EE for 24 h. The cells were harvested and whole-cell lysates were subjected to Western blot analyses. Ethanol was used as a vehicle control. Densitometric analyses of the bands were performed and ratios of Tra2β to β-actin are presented. Mean + SEM from three independent experiments are presented. *, Difference is significant, compared with vehicle control (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although a few studies have reported that estrogen regulates SR-BI and SR-BII expression in liver (4, 19, 20, 21), the mechanisms underlying estrogen regulation of SR-B alternative splicing remain unknown. In the present study, we demonstrate that estrogen, especially in the more potent form of synthetic estrogen, can cause SR-BI reduction and SR-BII elevation in a dose-dependent manner, doing so via differential regulation of the glycosylated and nonglycosylated forms of SR-BI and SR-BII. This study is also the first to show that estrogen regulates the splicing of the exogenously expressed SR-B minigene in hepatic cells, demonstrating a direct regulation of SR-B pre-mRNA alternative splicing by estrogen. Lastly, we identify ASF/SF2, Tra2{alpha}, and Tra2β as important splicing factors in the SR-B splicing machinery and demonstrate that estrogen regulates Tra2β expression in liver cells, indicating that estrogen may regulate SR-B alternative splicing through regulating Tra2β expression levels.

Our findings of overall down-regulation of SR-BI protein and up-regulation of SR-BII protein are similar to previous studies (4, 19, 20, 21). We observed that SR-BII was expressed at a relatively high baseline level in rat liver and HepG2 cells in our studies, which was inconsistent with the report by Graf et al. (19) but confirms the report by Mulcahy et al. (17). We did not observe a total loss of SR-BI with a dramatic gain in SR-BII levels after estrogen treatment in HepG2 cells, which was reported by Graf et al., but we did observe differential regulation of glycosylated and nonglycosylated SR-BI and SR-BII. SR-BI has been reported to be N-glycosylated (24, 25) and glycosylation is proposed to play a role in the folding and export of the SR-BI protein from the endoplasmic reticulum (24). SR-BII protein glycosylation and its significance have not yet been established. It is not known at this time whether the glycosylation of SR-BII is important for its stability, subcellular localization, or lipid transport activities. It is surprising that estrogen up-regulated the nonglycosylated form of SR-BII but not the glycosylated form. Posttranslational modification of SR-BII may be one of the factors in estrogen’s effect on liver cells. However, the significance of the estrogen regulation of glycosylated and nonglycosylated forms of SR-BII remains to be explored.

Both glycosylated and nonglycosylated SR-BI were down-regulated by estrogen. SR-BI is believed to function as a mediator of reverse cholesterol transport, thus protecting humans from the onset of atherosclerosis (11, 29, 30, 31). Interestingly, the results from the Heart and Estrogen/Progestin Replacement Study (32) and the Women’s Health Initiative studies (33) do not support the observational data that estrogen has beneficial effects against cardiovascular disease (18, 34, 35). The fact that estrogen down-regulates SR-BI and estrogen protects humans from cardiovascular disease seems to be paradoxical. However, estrogen up-regulation of SR-BII, a potentially more potent mediator for cholesterol efflux (17), also contributes to the overall effects of estrogen on cholesterol metabolism. Yet another consideration is that hepatic overexpression of SR-BI in mice significantly decreases plasma HDL-cholesterol levels (36, 37), and gene deletion or attenuation of SR-BI in mice results in substantially increased HDL-cholesterol levels (38, 39). These inverse effects of SR-BI on plasma HDL-cholesterol concentrations may be attributed to estrogen’s effect on HDL levels, which had been designated as a protection marker of cardiovascular disease. In addition, despite its effects on liver parenchymal cells, estrogen up-regulates SR-BI expression in liver Kupffer cells (21), and other studies demonstrated that SR-BI is a positive regulator of reverse cholesterol transport in macrophages (40). Thus, besides liver parenchymal cells, a broader examination of estrogen’s effects on other types of liver cells, mainly Kupffer cells, may be helpful to explain the overall significance of estrogen on cholesterol metabolism in liver.

The SR-BI and SR-BII protein expression changes after estrogen treatment could be due to protein stability alterations or SR-B pre-mRNA alternative splicing regulation. Based on the analyses of the SR-B minigene we constructed, the genomic region between exons 11 and 13 contains the required essential sequences for proper SR-B splicing, and partial deletions in the middle of introns 11 and 12 do not affect the functionality of the minigene. More importantly, estrogen caused SR-BI and SR-BII isoform ratio changes in HepG2 cells, demonstrating for the first time that estrogen has a direct effect on SR-B pre-mRNA splicing. Estrogen regulation of SR-B pre-mRNA splicing was also confirmed by its converse effect on MCF-7 breast cancer cells, in which SR-BI isoform expression was increased with estrogen treatment. This effect in MCF-7 cells has a reasonable and sound basis because estrogen, as well as increased SR-BI expression, can stimulate breast cancer cell proliferation (41), and the evidence showed a link between estrogen and SR-BI in MCF-7 cells (42, 43). These results suggest that estrogen can directly regulate SR-B mRNA splicing as well as modulate SR-B protein glycosylation. Whether the posttranslational modification impacts SR-B protein stability is not clear yet.

To gain insight into the underlying mechanism of estrogen regulation of SR-B splicing, we examined the splicing factors potentially involved in the SR-B splicing machinery. Generally, the splicing factors, including hnRNPs, SR proteins, and SR-related proteins, function as either essential or regulatory factors in the steps of spliceosome assembly when the pre-mRNA is spliced (27, 44). Our data showed that in HepG2 cells, exogenous overexpression of ASF/SF2, Tra2{alpha}, and Tra2β changed the splicing pattern of SR-B. Overexpression of Tra2β drastically enhanced exon 12 skipping, yielding higher levels of SR-BII, whereas depletion of endogenous Tra2β suppressed exon 12 skipping. These results indicate that the splicing factors ASF/SF2, Tra2{alpha}, and Tra2β, especially Tra2β, participate in SR-B splicing regulation. Our data also showed that estrogen up-regulated Tra2β expression in both rat liver and HepG2 cells. Taken together, Tra2β could potentially be the essential factor in the SR-B splicing machinery, and estrogen may cause SR-BI down-regulation and SR-BII up-regulation through induction of Tra2β expression.

ASF/SF2, an important member of the SR protein family of essential splicing factors, regulates alternative splicing in two ways: the selection of splicing sites and the activation of exonic splicing enhancers (45). The phosphorylation status of ASF/SF2 also plays a role in its activity regulation (46). Thus, although ASF/SF2 expression levels were not changed with estrogen treatment, it may be affected by estrogen in other ways. Human Tra2{alpha} and Tra2β, homologs of Drosophila sex determination factor transformer-2 gene product (47, 48), are the SR-like splicing factors demonstrating sequence-specific binding to the purine-rich enhancer sequences (49). Interactions between Tra2 proteins and other splicing factors also play an important role in the regulation of splicing (45, 50). Tra2β has been reported to activate or repress splicing in some human genes like SMN1 (51), clathrin light chain B (52), and {tau} (53, 54). How ASF/SF2, Tra2{alpha}, Tra2β, and possibly other factors may be involved in SR-B alternative splicing still awaits future exploration. Also, whether there is cooperation among the splicing factors is yet unknown. One of the important characteristics of these proteins is that they contain functional structures known as a RRM domain, which is a RNA-recognition motif, and RS domains, which are protein interaction domains (45, 55). In our studies, deletions of the Tra2β RS1, RS2, and RRM domains eliminated the effects of Tra2β on SR-B splicing, suggesting that both the RNA-binding function and protein interaction of Tra2β are required. For ASF/SF2, RRM domain deletion but not RS domain deletion reversed its regulation of SR-B splicing, suggesting that RNA binding is required for ASF/SF2 effects. Hence, the binding of these proteins and their corresponding elements in SR-B pre-mRNA, which are termed as exon and intron splicing enhancers and silencers (26), appear to be critical for their function in SR-B splicing.

In summary, our studies suggest that estrogen may regulate SR-B isoform expression at both the RNA splicing and posttranslational levels and that for alternative splicing regulation, estrogen may function through regulation of the expression of splicing factors ASF/SF2, Tra2{alpha}, and especially Tra2β. These studies provide in-depth insight into the molecular mechanisms of SR-B regulation by estrogen and a better understanding of estrogen’s role in HDL and cholesterol physiology, although further characterization is required.


   Footnotes
 
This work was supported by National Institutes of Health Grants HL078817 and HD35163 (to M.P.M.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 2, 2007

Abbreviations: ASF, Alternative splicing factor; E2, 17β-estradiol; EE, 17{alpha}-ethynyl estradiol; ER, estrogen receptor; FBS, fetal bovine serum; HDL, high-density lipoprotein; hnRNP, heterogeneous nuclear ribonucleoprotein; RRM, RNA recognition motif; RS, arginine-serine rich; SF2, splicing factor 2; siRNA, small interfering RNA; SR, arginine/serine-rich; SR-B, scavenger receptor class B; Tra, Transformer.

Received March 21, 2007.

Accepted for publication July 23, 2007.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
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