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Circulation Research. 2007;101:156-165
Published online before print June 7, 2007, doi: 10.1161/CIRCRESAHA.107.151746
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(Circulation Research. 2007;101:156.)
© 2007 American Heart Association, Inc.

Molecular Medicine

ATP-Binding Cassette Transporter A1 Gene Transcription Is Downregulated by Activator Protein 2{alpha}

Doxazosin Inhibits Activator Protein 2{alpha} and Increases High-Density Lipoprotein Biogenesis Independent of {alpha}1-Adrenoceptor Blockade

Noriyuki Iwamoto, Sumiko Abe-Dohmae, Makoto Ayaori, Nobukiyo Tanaka, Masatoshi Kusuhara, Fumitaka Ohsuzu, Shinji Yokoyama

From the Biochemistry Department (N.I., S.A.-D., N.T., S.Y.), Nagoya City University Graduate School of Medical Sciences; and the First Department of Internal Medicine (M.A., M.K., F.O.), National Defense Medical College, Tokorozawa, Japan.

Correspondence to Dr Shinji Yokoyama, Biochemistry, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan. E-mail syokoyam{at}med.nagoya-cu.ac.jp


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
ATP-binding cassette transporter A1 (ABCA1) is a rate-limiting factor for high-density lipoprotein (HDL) biogenesis. The ABCA1 gene expression is known to be upregulated by various transcriptional factors. However, negative regulation factors would be better targets for pharmacological modulation of HDL biogenesis. Doxazosin, an {alpha}1-adrenoceptor blocker, increased ABCA1 mRNA, its protein, and apolipoprotein A-I–mediated HDL biogenesis in THP-1 macrophages and CHO-K1 cells, independent of {alpha}1-adrenoceptor blockade. Analysis of the human ABCA1 promoter indicated that the region between the positions –368 and –147 that contains an activator protein (AP)2-binding site responsible for the effects of doxazosin. Overexpression of AP2{alpha} inhibited ABCA1 transcription in a dose-dependent fashion. Mutation in the AP2-binding site caused increase of the basal promoter activity and canceling both the transactivation by doxazosin and the trans-repression by AP2{alpha}. Doxazosin had no effect on ABCA1 mRNA level in HepG2 cells, which lack endogenous AP2{alpha}, and it reversed the inhibitory effect of AP2{alpha} expression in this type of cells. Chromatin immunoprecipitation and gel shift assays revealed that doxazosin reduced specific binding of AP2{alpha} to the ABCA1 promoter, as it suppressed phosphorylation of AP2{alpha}. Finally, doxazosin increased ABCA1 expression and plasma HDL in mice. We thus concluded that AP2{alpha} negatively regulates the ABCA1 gene transcription. Doxazosin inhibits AP2{alpha} activity independent of {alpha}1-adrenoceptor blockade and increases the ABCA1 expression and HDL biogenesis. AP2{alpha} is a potent pharmacological target for the increase of HDL.


Key Words: ABCA1 • doxazosin • HDL • AP2 • apoA-I


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
High-density lipoprotein (HDL) is a strong negative risk factor for atherosclerotic vascular disease.1,2 To support this finding, HDL removes cholesterol from the cells in culture. Cholesterol molecule is not catabolized in mammalian somatic cells and must be transported to the liver for its degradation to bile acids. HDL is thought to play a central role in this pathway and, thereby, also to remove cholesterol from the cells in premature atherosclerosis such as macrophage-derived foam cells. High concentrations of HDL may thus facilitate this reaction and prevent atherosclerotic cardiovascular diseases.

HDL is generated by interaction of cells with helical apolipoproteins such as apolipoprotein (apo)A-I.3 Familial HDL deficiency, Tangier disease, lacks this reaction in association with mutations in the gene of ATP-binding cassette transporter A1 (ABCA1),4–6 and the HDL-lowering drug probucol inhibits the reaction by inactivating ABCA1.7 Therefore, this reaction is thought to be mediated by ABCA1 as the main source of plasma HDL. Overexpression of ABCA1 in mice exhibited an increase of HDL and reduction of atherosclerosis,8,9 demonstrating that ABCA1 is a rate-limiting factor of HDL biogenesis and thereby a key factor for modulating atherosclerosis.

Expression of ABCA1 is regulated by transcriptional and posttranscriptional factors. Transcriptional regulation of ABCA1 is diverse, including pathways by several transcriptional factors and by various essential physiological molecules such as cAMP,10 tumor necrosis factor {alpha} or nuclear factor {kappa}B.11 Alternative transcription start sites are also known in various tissues.12 Among such regulators is the liver X receptor (LXR), which binds to the DR4 element of the ABCA1 proximal promoter13 and has been extensively investigated. Oxysterol is a strong endogenous ligand for LXR and thought to increase in parallel with cellular-free cholesterol. ABCA1 expression is indeed enhanced by cholesterol loading13 or by inhibiting acyl-CoA acyltransferase.14 Posttranscriptional regulation of ABCA1 takes place by calpain-mediated degradation, and interaction of ABCA1 with helical apolipoprotein interferes with this proteolytic regulation leading to "positive feedback" for HDL biogenesis.15,16

Transcription of ABCA1 is influenced by some pharmacological compounds that have reportedly increased plasma HDL in their clinical use. We found that fenofibrate, a peroxisome proliferator-activated receptor {alpha} agonist, increases ABCA1 transcription by an LXR-dependent manner,17 and verapamil, a calcium channel blocker, enhances the transcription by an LXR-independent mechanism.18

Doxazosin, an {alpha}1-adrenoceptor blocker, has been shown to raise plasma HDL cholesterol and decrease total or low-density lipoprotein (LDL) cholesterol and triglycerides in animals and humans.19–25 Cholesterol accumulation and atherosclerotic plaque formation in arterial walls were reportedly suppressed by doxazosin.26,27 We investigated the effect of doxazosin on HDL biogenesis by the ABCA1/apoA-I pathway. We found that the ABCA1 promoter activity was negatively regulated by activator protein (AP)2{alpha} and that doxazosin inhibited this interaction of AP2{alpha} by its dephosphorylation. This effect of doxazosin was independent of {alpha}1- adrenoceptor blockade.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Doxazosin mesylate, terazosin hydrochloride, phenoxybenzamine hydrochloride, TO-901317, and anti–{alpha}-tubulin antibody were purchased from Sigma. Rabbit and mouse anti-AP2{alpha} antibodies (SC-8975 and SC-25343), nonimmune control rabbit IgG and mouse IgG (SC-2027 and SC-2025), and protein A/G agarose (SC-2003) were from Santa Cruz Biotechnology. Anti–phospho-serine antibody was obtained from Chemicon Technology and anti-calnexin antibody was from BD Biosciences. Rabbit antiserum against ABCA1 was produced as described previously.15,28 ApoA-I was isolated from human plasma HDL as described previously.29 LDL was isolated from human plasma by ultracentrifugation.

Cell Culture
THP-1 cells (Health Science Research Resources Bank, Han-nan, Japan) were cultured in RPMI medium 1640 containing 10% FBS. The cells were differentiated by 3.2x10–7 mol/L phorbol 12-myristate 13-acetate (PMA) (Wako Pure Chemical) for 72 hours (THP-1 macrophages).30 CHO-K1 cells (American Type Culture Collection, Manassas, Va) were cultured in DF medium (1:1 mixture of DMEM and Ham’s F12 medium) with 10% FBS. HepG2 cells (American Type Culture Collection) were cultured in minimum essential medium Eagle {alpha} modification with 10% FBS. All cultures were in a humidified atmosphere of 5% CO2 and 95% air at 37C°.

RNA Extraction and Analysis
Total RNA was isolated using ISOGEN (Nippon Gene) and reverse-transcribed by SuperScriptIII (Invitrogen) with random oligonucleotide primers. Quantitative expression analysis was performed in an ABI PRISM 7700 (Applied Biosystems) using SYBR Green technology. The following primers were used in polymerase chain reaction (PCR) (forward and reverse, respectively): ABCA1 messages, 5'-GCTTTCAATCATCCCCTGAA-3' and 5'-CAGGTGTT TGCTTTGCTGA-3' for human and 5'-AAGGGGTGGTGTTC TTCCTC-3' and 5'-CCTCACATCCTCATCCTCGT-3' for hamster; glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 5'-GACCTGCCGTCTAGAAAAACC-3' and 5'-ACCACCTGGTGCTCAG-TGTAG-3' for human and 5'-CCATGGAGAAGGCCGGG-GCCC-3' and 5'-AAGGGGTGGTGTTCTTCCTC-3' for hamster; AP2{alpha}, 5'-GATCCTCGCAGGGACTACAG-3' and 5'-TACCCGGGTCTTCTACATGC-3'. The results were normalized for GAPDH. For Northern blotting, 15 µg of total RNA was separated by electrophoresis using a 1% (wt/vol) agarose/formaldehyde gel and transferred to a nylon membrane. The membranes were hybridized with the radio-labeled probes with [{alpha}-32P]dCTP using a Random Prime DNA Labeling Kit (Takara Bio, Otsu, Japan). Probes for human ABCA1 were constructed from cDNA fragments obtained from THP-1 cells as a template by using RT-PCR.31

Reporter Gene Assay
The human ABCA1 promoter region containing the nucleotides –2245 to +110 was derived from the human BAC clone using a template kindly provided by Dr H. Bryan Brewer Jr. (National Heart, Lung, and Blood Institute, Bethesda, Md). For the luciferase reporter assay, ABCA1 promoter constructs were generated by PCR using specific primers with HindIII sites. Each PCR product was ligated into the HindIII site of pGL3 Basic vector (Promega, Madison, Wis). To introduce mutations in 5' half-site of the ABCA1 DR4 element (DR4m) and AP2-binding site (AP2m), site-directed mutagenesis was introduced using a Quick Change II Site-Directed Mutagenesis kit (Stratagene La Jolla, Calif) using the following primers (lower-case letters indicate mutated nucleotides, underlines indicate transcription factors binding site): DR4, 5'-CGAGCGCAGA-GGTTACTATCtGcagAAGCCTGTGCTCTCCC-3', 5'-GGGAGA-GCACAGGCTTctgCaGATAGTAACCTCTGCGCTCG-3'31; and AP2, 5'-CCGCGCGTCTTAGGCtGGaGGGCtCGtGCGGGGGAA-GGG-3' and 5'-CCCTTCCCCCGCaCGaGCCCtCCaGCCTAAG-ACGCGCGG-3'. The plasmid for expression of pSG5-neo-AP2{alpha}, its alternatively-spliced gene product AP2{alpha}A (AP-2B32) as a negative control, and the control vector32 were kindly provided by Dr M. A. Tainsky (Wayne State University, Detroit, Mich). To analyze promoter activity of ABCA1, cells were subcultured in a 24-well tray and incubated for 24 hours. The reporter luciferase plasmids (1 µg) and the phRL-TK (Promega) encoding Renilla luciferase (for normalization; 30 ng) were transfected by using Lipofectamine 2000 (Invitrogen) for CHO-K1 and jet-PEI Man (Polyplus transfection) for THP-1 macrophage. Expression plasmid of AP2{alpha}, AP2{alpha}A, or the empty vector (mock) was cotransfected, and the cells were incubated for 48 hours. Doxazosin or other compounds was present for the last 16 hours, the cells were lysed, and the luciferase activity was measured using the Dual-Luciferase Reporter system (Promega).

Western Blotting Analysis for ABCA1 and Chromatin Immunoprecipitation Assay
Immunoblotting of ABCA1 was performed as previously described by using specific polyclonal antibody.15,28 The chromatin immunoprecipitation assay was performed as described previously.33 Human ABCA1 promoter containing the AP2-binding site was amplified with specific primers: 5'-CGGGAACGTGGACTAGAGAG-3' and 5'-TGGAGGGTACAGCAGGTGTC-3'.

Gel Shift Assay
The AP2{alpha}-transfected CHO-K1 cells were incubated for 48 hours, and the nuclear extract was prepared. A double-strand DNA fragment of human ABCA1 promoter –279 to –317 was labeled at the 3' end with digoxigenin-11-ddUTP by using a DIG Gel Shift Kit (Roche Applied Science). The electrophoresis bands were visualized with an anti-digoxigenin antibody conjugated with alkaline phosphatase.

RNA Interference
Small interfering (si)RNA specific for human AP2{alpha} and a scramble control were obtained from Invitrogen and transfected into THP-1 cells using Amaxa transfection machine (Amaxa Inc) according to the protocol of the manufacture. Immediately after transfection, THP-1 cells were differentiated for 16 hours, and specific mRNA expression was analyzed by quantitative RT-PCR.

Cellular Lipid Release
THP-1 macrophages were incubated with apoA-I, 10 µg/mL, in RPMI medium 1640 containing 0.1% BSA for 16 hours, and cholesterol and choline-phospholipid in the medium were enzymatically measured.34

In Vivo Experiments
Doxazosin, 25 mg/kg per day, was given to 10-week-old male C57BL/6 mice for 1 week using a gastric tube. Blood was collected by cardiac puncture under anesthesia. Animals were euthanized by cervical dislocation, and the livers were removed immediately for quantitation of mRNA by RT-PCR and ABCA1 protein by Western blotting. Plasma HDL was analyzed by agarose gel electrophoresis with Sudan Black staining by high-performance liquid chromatography (HPLC) by monitoring total and free cholesterol, choline phospholipid, and triacylglycerol35 at Skilight Biotech (Akita, Japan) and by ultracentrifugation. The experimental protocol was preapproved by the institutional committee for animal welfare.

Statistical Analysis
Statistical analysis was performed by 1-way ANOVA followed by Scheffé’s test. All values represent average±SD.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Doxazosin Increases ABCA1 and HDL Biogenesis
Doxazosin increased the apoA-I–mediated release from THP-1 macrophage of cholesterol and phospholipid (Figure 1A) and induced ABCA1 protein expression (Figure 1B) both in a dose-dependent manner. The increase of ABCA1 by doxazosin was not influenced by cotreatment of the cells with phenoxybenzamine, an irreversible blocker of {alpha}1- and {alpha}2-adrenoceptors (Figure 1C). Increase of ABCA1 was not apparent by phenoxybenzamine or terazosin, another selective {alpha}1-adrenoceptor blocker (Figure 1D). Terazosin did not increase the lipid release in the same condition (data not shown). Thus, doxazosin increases ABCA1 protein and HDL biogenesis independent of the {alpha}1-adrenoceptor pathway.


Figure 1
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  		Figure 1. Doxazosin increases ABCA1 protein and HDL biogenesis. A, THP-1 macrophages were incubated in medium containing 0.1% BSA for 16 hours with the indicated concentration of doxazosin in the presence and absence of 10 µg/mL apoA-I. When cotreated with phenoxybenzamine, it was added 1 hour before the incubation with doxazosin. Cellular lipid release was determined by measuring lipid in the medium with the enzymatic assay. B through D, THP-1 macrophages were treated with doxazosin as above in the absence of apoA-I. ABCA1 protein was analyzed by Western blotting. Numbers below the gels indicate the results of scanning ABCA1 bands standardized for references. Graph data represent averages±SD for triplicate assays. *P<0.05, **P<0.01, ***P<0.001 vs the data without doxazosin with 10 µg/mL apoA-I.

Doxazosin Increases ABCA1 mRNA Expression Level
ABCA1 mRNA was increased by doxazosin in a dose-dependent manner in THP-1 macrophages, as shown by Northern blotting analysis (Figure 2A) and by quantitative RT-PCR (Figure 2B). Neither terazosin nor phenoxybenzamine affected ABCA1 mRNA. The effect of doxazosin was not interfered by coexposure of the cells to phenoxybenzamine (Figure 2C). Increase of ABCA1 mRNA was also demonstrated in CHO-K1 cells (Figure 2D). When ABCA1 transcription was increased through LXR by loading cholesterol or by its agonist, TO901317, doxazosin further increased the mRNA (Figure 2E). Transfection of DNA plasmids to THP-1 macrophages was technically difficult to yield consistent efficiency; therefore, we used CHO-K1 cells for the ABCA1 promoter analysis thereafter.


Figure 2
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  		Figure 2. Effects of {alpha}1-adrenoceptor blockers on the expression of ABCA1 mRNA. Expression of the ABCA1 mRNA was analyzed by Northern blotting (A) and quantitative RT-PCR (B, C, and D) in THP-1 macrophages (A, B, and C) and CHO-K1 (D) cells. All compounds were incubated with the cells for 16 hours. Message of ABCA1 was standardized for that of GAPDH. When cotreated with phenoxybenzamine, it was added 1 hour before the incubation with doxazosin (the right-most columns in C and D). The effect of doxazosin was examined as ABCA1 transcription was upregulated in THP-1 macrophages, by loading LDL (150 µg/mL) or by LXR ligand TO901317 (E). Numbers below the gels indicate the results of scanning of ABCA1 bands standardized for references. Data points represent means±SD for triplicate assays. **P<0.01 and ***P<0.001 vs the controls (no doxazosin).

ABCA1 Promoter Analysis
To investigate molecular mechanism for doxazosin to upregulate ABCA1, we prepared deletion constructs for the promoter region between –940 and +110 bp (WT1 to WT5) and a mutant construct of the LXR-binding element (DR4m) (Figure 3A). Transcriptional enhancement by doxazosin of human ABCA1 gene was examined by using the reporter gene WT1 (Figure 3B). Doxazosin significantly increased the promoter activity regardless of inhibition of {alpha}1-adrenoceptor by phenoxybenzamine. Promoter activities of WT1, WT2, and WT3 of ABCA1 were increased by doxazosin in a dose-dependent manner, but those of WT4 and WT5 were not influenced (Figure 3C). With the DR4m construct, luciferase activity was also increased by doxazosin (Figure 3C). The results thus indicated that the effect of doxazosin is independent of the LXR pathway and requires the region between –368 (WT3) and –147 (WT4).


Figure 3
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  		Figure 3. Analysis of the ABCA1 promoter for the reactivity to doxazosin. A, Schematic diagram of the various ABCA1 promoter constructs for the reporter assay. B, THP-1 macrophage were transfected with the ABCA1 promoter constructs WT1 from –940 to +110. After 2 days of transfection, cells were incubated with doxazosin or phenoxybenzamine for 16 hours. Luciferase activity was measured and normalized for phRL-TK. C, Each of the reporter constructs was transfected in CHO-K1 cells and incubated with doxazosin or LXR agonist (TO901317) for 16 hours. Luciferase activity was expressed as a relative value to that in control cells for each construct. Data represent means±SD for 3 independent experiments. **P<0.01 and ***P<0.001 vs the control.

AP2{alpha} Negatively Regulates ABCA1 Promoter
Doxazosin reportedly inhibited cell proliferation by stabilizing cell cycle at the G0/G1 phase36 and induced apoptosis of benign and malignant prostate cells.37,38 The doxazosin-reactive ABCA1 promoter region between –368 and –147 contains the AP2-binding site, and AP2 is thought to be associated with apoptosis, cell proliferation and differentiation.39 Therefore, luciferase assay for the ABCA1 promoter was attempted in the cells in which AP2{alpha} or AP2{alpha}A (an inactive mutant) is overexpressed (Figure 4A). AP2{alpha}, but not AP2{alpha}A, downregulated ABCA1 promoter activity in a dose-dependent manner. When the AP2-binding site was mutated (AP2m; Figure 3A), the basal luciferase activity was higher than WT1 and did not change by overexpression of AP2{alpha} (Figure 4B). Doxazosin increased the ABCA1 promoter activity of WT1 but not that of AP2m (Figure 4C).


Figure 4
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  		Figure 4. AP2{alpha} negatively regulates the ABCA1 promoter activity. The ABCA1 promoter activity was measured in CHO-K1 (A, B, and C) and HepG2 (D) cells in 24-well plates. Luciferase activity was analyzed as described in Figure 1. A, CHO-K1 cells were transfected with WT1 (Figure 2A) reporter gene and AP2{alpha} or its negative control AP2{alpha}A expression vector in the indicated amount of DNA per well. B, WT1 or AP2m (Figure 2A) constructs and 250 ng of AP2{alpha} plasmid were transfected in CHO-K1. C, WT1 and AP2m were transfected and incubated with or without doxazosin for 16 hours. D, HepG2 cells, which lack AP2{alpha}, were transfected with WT1 and 250 ng of AP2{alpha} plasmid. E and F, AP2{alpha} expression was knocked down by siRNA in THP-1. After transfection of siRNA, the cells were differentiated into macrophages for 2 days by incubation with PMA. After 3 days, doxazosin was added to the medium and incubated for 16 hours. Total RNA was isolated from the cells, and relative mRNA levels of AP2{alpha} (E) and ABCA1 (F) were measured by quantitative RT-PCR analysis. Data were normalized to GAPDH levels. The values represent the average±SD relative to the untreated cells (scramble control and no doxazosin) from 2 independent experiments performed in triplicate. Significance of difference is indicated as *P<0.05, **P<0.01, and ***P<0.001 vs WT1- and mock-transfected cells (B) vs WT1-transfected cells with 0 µmol/L doxazosin (C) for the indicated pairs (D) and vs the untreated cells (E and F).

Doxazosin had no effect on the ABCA1 promoter activity in HepG2 cells, which lack AP2{alpha}.40 The luciferase activity in HepG2 cells was reduced by transfection of AP2{alpha}, and this effect was reversed by doxazosin (Figure 4D).

In THP-1 macrophages, AP2{alpha} mRNA expression was only slightly decreased by doxazosin and significantly downregulated by a specific siRNA to <20% (Figure 4E). In the AP2{alpha}-knockdown condition, ABCA1 mRNA level was somewhat higher than the scramble control, and doxazosin caused change of the ABCA1 mRNA to a lesser extent than in the control (Figure 4F). These data demonstrated that AP2{alpha} negatively regulates the ABCA1 promoter activity and doxazosin interferes with the AP2{alpha} interaction with the ABCA1 promoter.

Doxazosin Decreases Binding of AP2{alpha} to the ABCA1 Promoter
To investigate the AP2{alpha} interaction with the ABCA1 promoter, chromatin immunoprecipitation assay was performed. AP2{alpha} binds to the AP2-binding site in the ABCA1 promoter region, and the complex formation was reduced by doxazosin in a dose-dependent manner (Figure 5A). Western blotting analysis revealed that doxazosin did not change total AP2{alpha} protein level in this condition (Figure 5B left panel). As transcription activity of AP2 was reportedly modulated by serine phosphorylation,41,42 change in phosphorylation of AP2{alpha} by doxazosin was examined. Figure 5B demonstrates that AP2{alpha} was dephosphorylated by doxazosin dose dependently in both THP-1 macrophage and CHO-K1 cells. The expression level of total AP2{alpha} was also reduced by doxazosin in CHO-K1 but not in THP-1 macrophages. Terazosin and phenoxybenzamine did not cause such an effect on phosphorylation (Figure 5C). Gel-shift assay confirmed the AP2{alpha} binding to the putative AP2-binding site in vitro (Figure 5D). The DNA–protein complex was found in the presence of recombinant AP2{alpha} but not with AP2{alpha}A. Incubation with an anti-AP2{alpha} antibody caused supershift of the DNA–protein complex band. Competition experiments with the unlabeled DNA probe and its mutated probe showed that the interaction is at the AP2-binding site. In the THP-1 macrophages, DNA–protein binding was decreased by doxazosin.


Figure 5
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  		Figure 5. Doxazosin increases ABCA1 promoter activity through decline of DNA binding of AP2{alpha} by its dephosphorylation. A, THP-1 macrophages were incubated with the indicated concentrations of doxazosin for 16 hours. Each sample was immunoprecipitated with an anti-AP2{alpha} antibody or normal IgG. DNA was eluted and purified. PCR was performed by using the specific primers for the ABCA1 promoter region containing AP2{alpha}-binding sites. B and C, After 16 hours of incubation with doxazosin or other {alpha}1-adrenoceptor blockers, cells were lysed and immunoprecipitated with an anti-AP2{alpha} antibody. Western blotting analysis was performed to detect phosphorylated AP2{alpha} by using phospho-serine–specific antibody. D (left), The results of the gel shift assay for the nuclear extract of the AP2{alpha}-transfected CHO-K1 cells as described in the text. Anti-AP2{alpha} antibodies SC-8975 (rabbit) and SC-25343 (mouse) are indicated as (r) and (m), respectively, as well as for control IgGs. D (right), The gel-shift assay for the doxazosin-treated THP-1 macrophages. Numbers below the gels indicate the results of scanning of ABCA1 bands standardized for references.

Increase of HDL-Cholesterol by Doxazosin In Vivo
Effects of doxazosin were examined in mice for ABCA1 and plasma HDL. The mRNA and protein of ABCA1 were both increased in the liver by the doxazosin treatment (Figure 6AB). Electrophoretic analysis of plasma showed increase of HDL (Figure 6C). HPLC analysis of plasma confirmed the increase of HDL, demonstrated by elution profiles of lipid (Figure 6D), and lipid quantitation for each lipoprotein fraction (Table). Lipid increase was also observed in LDL to a less extent. Change in HDL size was not obvious. Ultracentrifugal analysis confirmed significant increase of plasma HDL cholesterol by doxazosin (data not shown).


Figure 6
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  		Figure 6. Effect of doxazosin in C57BL/6 mice. The doxazosin-treated and control mice (n=5 each) were analyzed for ABCA1 expression in the liver and plasma HDL. The mice were treated with doxazosin as described in the text. The liver tissue (30 mg wet weight) was homogenized. Total RNA (50 ng) was used for quantitative RT-PCR for ABCA1 mRNA (A), and 50 µg of protein was analyzed by Western blotting for ABCA1 protein (B). Numbers below the gels indicate the results of scanning of ABCA1 bands standardized for references. Blood plasma was analyzed by electrophoresis in agarose gel and lipid staining with Sudan black (C) and by HPLC by monitoring total and free cholesterol, choline phospholipid, and triacylglycerol (D). *P<0.05.


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  		Table 1. Plasma Lipoprotein–Lipid Concentration in Mice


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
We demonstrated that human ABCA1 gene was negatively regulated by AP2{alpha}. Doxazosin dephosphorylates AP2{alpha} and suppresses its interaction with the ABCA1 promoter. This causes transcriptional upregulation of the ABCA1 gene and increases HDL biogenesis. Increase of ABCA1 and plasma HDL was confirmed in vivo in mice. Other {alpha}1-adrenoceptor inhibitors were ineffective; therefore, this effect is unlikely related to inhibition of {alpha}1-adrenoceptor.

AP2{alpha} is a sequence-specific DNA-binding transcription factor that functions in differentiation and development. It interacts with many other transcriptional factors39 such as Rb43,44 and regulates the cell cycle.44 Doxazosin reportedly induced cell cycle arrest at the position from the G0/G1 phase to S phase by inhibiting p27 downregulation and preventing mitogen-induced Rb hyperphosphorylation in human smooth muscle cells.36 It was thus analyzed whether AP2{alpha} affects the ABCA1 promoter activity through interaction with Rb. Rb was also dephosphorylated by doxazosin, but Rb did not influence the ABCA1 promoter activity regardless of cotransfection with AP2{alpha} (see the online data supplement at http://circres.ahajournals.org). The doxazosin-activated ABCA1 promoter region between –368 and –147 contains binding sites for other transcriptional factors, such as zinc finger gene 202 (ZNF202) and SP1. ZNF202 has been identified as a transcriptional repressor of various genes involved in lipid metabolism, such as apolipoproteins A-IV, C-III, and E, phospholipid transfer protein, and hepatic nuclear factor 4,45 as well as ABCA1 and ABCG1.46 Doxazosin in fact reduced the expression of ZNF202 (data not shown). SP1 is an important component of the eukaryotic cellular transcriptional machinery. AP2 interacted with SP1 and blocked its stimulatory function in some gene-binding sites.47 Thus, the mechanism for doxazosin to interfere with the function of AP2 may involve additional transcriptional factors.

Doxazosin reduces phosphorylation of AP2{alpha} (Figure 5BC). Therefore, we examined phosphorylation of Erk1/2 and Akt/protein kinase B (PKB), which are downstream signals of growth factor receptors. Doxazosin significantly reduced the Akt/PKB phosphorylation in THP-1 macrophage (data not shown), which is consistent with the previous findings that induction of apoptosis by doxazosin can be partly attributed to inhibition of Akt/PKB activation.48 We further examined whether doxazosin inhibits growth factor receptors but yielded inconclusive results on the dephosphorylation of Akt/PKB and Erk1/2 by platelet-derived growth factor, insulin, or epidermal growth factor (data not shown).

The findings in this study provide us new background and strategy for technology to raise plasma HDL. The results also provided the molecular evidence to support the clinical finding that doxazosin increases plasma HDL.19–25 The clinical benefit of doxazosin itself, however, is controversial. Cardiovascular risk increased by doxazosin in comparison to diuretics,49 which may be attributed to its induction of apoptosis in cardiomyocytes.50 It is unclear whether this is related to its AP2{alpha} inhibition. Increase of plasma HDL was accompanied by small increase of LDL in mice, which is consistent with the findings in ABCA1 transgenic mice.8,9 The results of the in vivo study in mice are still preliminary. Further studies are required for clinical application of ABCA1 upregulation by AP2{alpha} inhibition.


*    Acknowledgments
 
Sources of Funding

This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Japan Health Science Foundation and by the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (Japan).

Disclosures

None.


*    Footnotes
 
Original received January 30, 2007; resubmission received March 8, 2007; revised resubmission received May 3, 2007; accepted May 24, 2007.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Gordon DJ, Rifkind BM. High-density lipoprotein–the clinical implications of recent studies. N Engl J Med. 1989; 321: 1311–1316.[Medline] [Order article via Infotrieve]
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Old Drug, New Tricks: The Unexpected Effect of Doxazosin on High-Density Lipoprotein
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Old Drug, New Tricks: The Unexpected Effect of Doxazosin on High-Density Lipoprotein
Circ. Res., July 20, 2007; 101(2): 116 - 118.
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