Identification of the cAMP-Responsive Enhancer of the Murine ABCA1 Gene
Requirement for CREB1 and STAT3/4 Elements
Objective— To determine the mechanism by which expression of the murine ABCA1 gene is highly induced by cAMP analogues.
Methods and Results— ABCA1 mRNA turnover cannot account for its induction by cAMP. Thus cAMP induction of ABCA1 mRNA occurs at a transcriptional level. Shotgun cloning DNA fragments from the murine ABCA1 locus identified a strong cAMP responsive enhancer located in the first intron, which led to 25- to 100-fold cAMP-mediated induction of reporter gene activity. Deletions and mutations of this enhancer led to the identification a cAMP-responsive element (CRE) that was essential for the cAMP induction. Furthermore, the capacity of this CRE site to mediate the cAMP induction required the presence of a STAT3/4 element located 81 bp away. A dominant-negative CREB expression vector inhibited the cAMP induction of ABCA1, demonstrating that CREB was required for cAMP induction of ABCA1 expression in RAW264.7 cells.
Conclusion— Phospho-CREB1 controls the cAMP-mediated induction of murine ABCA1 gene expression through a CRE site acting in cooperation with a nearby STAT element. This CRE site is not conserved in the human ABCA1 gene, explaining why human ABCA1 is not strongly stimulated by cAMP analogs.
ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-poor high-density lipoprotein (HDL) apolipoproteins and plays a key role in the elimination of cholesterol from macrophage-derived foam cells in the artery wall.1 Tangier disease, which is characterized by very low HDL levels, cholesterol deposition in macrophages, and premature atherosclerosis, is caused by mutations in ABCA1 gene.2–4 Macrophage ABCA1 has been shown to protect against atherosclerosis in mouse models, as transplantation of ABCA1-deficient marrow, compared with transplantation of wild-type marrow, resulted in an increase of atherosclerosis in apolipoprotein (apo) E-deficient mice.5,6 Thus, modulation of ABCA1 expression in macrophages constitutes a therapeutic target for the prevention of human atherosclerosis.
Although ABCA1 is highly regulated at a post-translational level,7–9 ABCA1 gene expression is also tightly regulated in macrophages. ABCA1 can be activated by the nuclear liver X receptor and retinoic X receptor in response to oxysterols or synthetic ligands,10,11 by PPAR-α and PPAR-γ activators,12 and by retinoic acid receptor ligands.13
ABCA1-mediated cholesterol efflux to apolipoprotein AI (apoAI) is stimulated by cAMP analogs in mouse macrophages14,15 as a result of &50- to 70-fold induction of ABCA1 gene expression.16,17 However, the response to cAMP differs between mouse and human ABCA1 genes,18 and among various cell lines tested, only mouse macrophages show a substantial induction of ABCA1 mRNA and cholesterol efflux by a cAMP treatment.19 In vitro promoter analysis suggests that the proximal promoter of the human ABCA1 gene contains element(s) responsible for &2-fold cAMP-mediated induction of luciferase reporter gene activity in RAW264.7 cells.20 However, the elements involved in this modest cAMP-mediated induction cannot account for the &50- to 70-fold increase of ABCA1 mRNA observed in RAW264.7 cells by a treatment with cAMP.17
In this study, we conclude that cAMP-mediated induction of ABCA1 occurs at a transcriptional level in the mouse macrophage RAW264.7 cell line. We identified a consensus cAMP response element (CRE) in the first intron of the murine ABCA1 gene that mediated cAMP induction of reporter gene constructions in cooperation with a nearby STAT3/4 element. We further report that the activated form of CRE-binding protein (CREB), ie, phospho-CREB, specifically binds the CRE in the first intron of ABCA1 gene, and that use of a dominant-negative CREB inhibits the cAMP induction of reporter transcripts mediated by this CRE. The dominant-negative CREB also reduced ABCA1 levels in cAMP-treated RAW264.7 cells, confirming that CREB is required for cAMP-mediated induction of murine ABCA1.
A 1131-bp DNA fragment corresponding to the −1104/+26 region of murine ABCA1 gene promoter was amplified by polymerase chain reaction (PCR) and subcloned using the TA overhang into the pCR2.1 vector (Invitrogen). The sequences of both upstream and downstream primers used for the generation of all constructs by PCR are presented in Table I (available online at http://atvb.ahajournals.org). Then, a 1189-bp HindIII fragment containing the ABCA1 promoter and a portion of the pCR2.1 polylinker was isolated and cloned into the HindIII-cut pGL3-Basic vector (Promega), generating the pABCA1 construct (luciferase expression vector driven by the 1.1-kb murine ABCA1 promoter). The orientation and the integrity of the insert were verified by sequencing. Generation of constructs by shotgun cloning from the BAC RPCI 23-25 D17 are described in the supplementary materials (please see http://atvb.ahajournals.org).
The constructs pCMV500 and A-CREB, expressing a control plasmid and the dominant-negative inhibitor of CREB, respectively,21 were the generous gifts of Dr Charles Vinson (National Cancer Institute, NIH, Bethesda, Md).
RAW264.7 cells were treated in the presence or absence of either 0.3 mmol/L 8Br-cAMP or 4 μg/mL 22(R)-hydroxycholesterol and 1 μmol/L 9-cis-retinoic acid in DGGB for desired time, as indicated in the figure legends. Total RNA was extracted with TRIzol reagent (Gibco BRL) and 1 μg of RNA was reverse-transcribed into cDNA using Superscript II (Gibco BRL). PCR amplification and normalization were performed as previously described.17
Cholesterol Efflux Assays
Lipid efflux to apoAI (Biodesign, Saco, Me) from cholesterol-loaded RAW264.7 cells was performed as previously described.22 The percentage of cholesterol efflux was calculated as 100 ×(medium dpm)/(medium dpm+cell dpm).
Please see the expanded Methods section at http://atvb.ahajournals.org for transfection experiments, electrophoretic mobility shift assays, and chromatin immunoprecipitation analysis.
Western Blot Analysis
RAW264.7 cells were grown in 6-well plates to 80% confluence and incubated in DGGB in the presence or absence of 0.3 mmol/L 8Br-cAMP for 16 hours. Detection of ABCA1 by Western blot analysis was performed as previously described.7
Data are shown as mean±SD. Comparisons of 2 groups were performed by a 2-tailed t test, and comparisons of 3 or more groups were performed by ANOVA with Dunnett’s posttest. All statistics were performed using Prism software from GraphPad (San Diego, Calif).
cAMP Induces ABCA1 Expression at a Transcriptional Level
To address the mechanism by which cAMP induces the expression of the murine ABCA1 gene, we analyzed the specificity of the 8Br-cAMP–mediated stimulation of ABCA1 mRNA by reverse-transcription PCR in the murine RAW264.7 macrophage cell line. As shown in Figure 1A, 8Br-cAMP is the most potent inducer of ABCA1 mRNA expression in RAW264.7 cells as compared with acetylated low-density lipoprotein (acLDL) or 22-hydroxycholesterol (22OHC) and 9-cis retinoic acid (9cRA) (55-fold versus 3.3- and 4.5-fold, respectively), and such an induction is rapidly observed after only a 2-hour treatment (&30-fold) (Figure 1B). Addition of either acLDL or 22OHC and 9cRA to 8Br-cAMP was accompanied by a synergistic increase of ABCA1 mRNA (Figure 1A), indicating that those inducers, which act via the LxR transcription factor, and cAMP mediate ABCA1 induction by distinct mechanisms.
To determine whether cAMP-mediated ABCA1 mRNA induction results from an increase of mRNA stability, we analyzed the effects of 8Br-cAMP on ABCA1 mRNA turnover. Analysis of ABCA1 mRNA for 6 hours after the addition of actinomycin D indicated that ABCA1 mRNA turnover was faster in the 8Br-cAMP–treated cells with a half-life of 1.9 hours compared with 3.5 hours in 22OHC plus 9cRA-treated cells (Figure 1C). Because the levels of ABCA1 mRNA were increased by 8Br-cAMP and ABCA1 mRNA was not stabilized by 8Br-cAMP, we conclude that 8Br-cAMP induced ABCA1 expression at a transcriptional level.
Identification of cAMP-Responsive Regions in the ABCA1 Gene
To identify cAMP response elements (CRE) in the mouse ABCA1 gene, a bioinformatics approach was tried but was not successful (data not shown). Then a shotgun cloning strategy was performed by cloning restriction fragments derived from a mouse ABCA1 gene region BAC clone upstream of the 1.1-kb proximal mouse ABCA1 promoter driving a luciferase reporter gene. Among the 41 constructs tested in transient transfection experiments in RAW264.7 cells, luciferase activities of construct numbers 2, 6, 26, 31, and 37 were substantially induced by a 16-hour treatment with 0.3 mmol/L 8Br-cAMP compared with the control pABCA1 construct (Figure I, available online at http://atvb.ahajournals.org).
Restriction mapping with EcoRI and DNA sequencing of the insert ends revealed that the same 3.6-kb DNA fragment upstream of the ABCA1 gene (fragment A) was inserted in constructs 2 and 31, and that constructs 26 and 37 (the most highly induced constructs) shared the same 2.2-kb DNA fragment from intron 1 (fragment B). A larger 8.7-kb DNA fragment of intron 5 (fragment C) was the insert in construct 6 (Figure 2A). Thus the 5 active constructs were caused by only 3 different inserts, A, B, and C, which each contain a DNA element that mediated induction by cAMP. On performing independent transfection experiments to compare the inducing strength of the A, B, and C fragments (Figure 2B), fragment B consistently yielded the highest induction of luciferase activity by 8Br-cAMP (74-fold, P<0.01 in Figure 2B), whereas fragments A and C mediated modest inductions of luciferase activity by 8Br-cAMP (&4 and &7-fold, respectively). However, we noticed that the cAMP-mediated induction of fragment B varied from experiment to experiment and ranged from &25 to >100-fold. Thus fragment B, located in the first intron of ABCA1 gene, contained a strong cAMP-responsive element that may account for the majority of the &70-fold induction of ABCA1 mRNA by cAMP.
CRE on B Fragment Is Required for cAMP-Mediated Activation
To identify the specific cAMP-responsive element(s) in the B fragment, a series of deletion constructs were made and analyzed via transient transfection. As shown in Figure 3A, the construct that contains the full-length B fragment (the B sequence in Figures 3 and 4⇓ is inverted relative to the coding strand of mouse ABCA1 gene, and thus corresponds to the noncoding strand) displayed a significant 36-fold induction of luciferase activity (P<0.01) by 8Br-cAMP as compared with the pABCA1 parent construct (the luciferase expression vector driven by the 1.1-kb murine ABCA1 promoter). Deletion of a 1.2-kb from the 5′ end of the B insert (B-2) completely abolished the 8Br-cAMP induction, whereas a 5′ 888 bp deletion (B-1) was still cAMP-inducible, suggesting that the active element resided within the 320-bp region at the 5′ end of the B-1 fragment (this 320-bp region is called fragment b). Surprisingly, a construct that only contained the b sequence was only weakly inducible by 8Br-cAMP. However, a longer fragment (b′), including a 46-bp extension at the 3′ end of the b fragment, restored most of the activity found in the B fragment (25-fold induction by 8Br-cAMP; P<0.01), suggesting that some additional elements in the b′ construct were required.
Sequence analysis of the B-1 and b′ constructs identified a consensus CRE (5′-TGACGTCC-3′), within which was found the AatII restriction site that was used to generate the B-2 deletion fragment. Thus the B-2 fragment contained only a truncated CRE site. The b fragment also did not contain the intact CRE site. We hypothesized that a single CRE located at the AatII site in the B fragment was required for the cAMP-dependent enhancer activity of this fragment. To test this hypothesis, AatII digestion, overhang elimination, and blunt end ligation was used to create 4-bp deletions (5′-TGCC-3′) in the CRE of B and b′, (constructs B-Mut and b′-Mut, respectively). As expected, luciferase activities of the B-Mut and b′-Mut constructs were not significantly induced by 8Br-cAMP (Figure 3A), thus confirming that this CRE was necessary for cAMP induction.
Phospho-CREB1 Specifically Binds the B Fragment CRE
Gel mobility shift assays were performed to verify whether the activated form of CREB1, ie, phospho-CREB1, binds to the CRE identified in the B fragment. As shown on Figure 3B, incubation of a biotinylated probe (CRE-b′) with nuclear extracts of control-treated RAW264.7 cells did not lead to the formation of any DNA-protein complexes (Figure 3B, lanes 2 to 6), whereas 2 complexes (I and II) were observed with nuclear extracts of 8Br-cAMP–treated RAW264.7 cells (lane 7). Both complexes were also observed using a biotinylated probe specific for the consensus sequence of the CREB/ATF-transcription factor family (CREB, lane 15). A 4-bp deletion in the biotinylated probe (CRE-b′Mut), corresponding to the mutation that neutralized cAMP induction mediated by the B fragment (Figure 3B), prevented the formation of the 2 complexes with nuclear extracts of 8Br-cAMP–treated RAW264.7 cells (lane 14). Finally, the use of an antibody against phospho-CREB1 (phospho-Ser 133) confirmed that complex I was formed as a result of interactions with phospho-CREB1 (lanes 10 and 18).
The binding of phospho-CREB1 on the b′ CRE in situ was investigated by chromatin immunoprecipitation (ChIP) analysis using RAW264.7 cells treated for 16 hours with or without 0.3 mmol/L 8Br-cAMP. As shown on Figure 3C, overnight incubation of protein–DNA complexes with an antibody raised against phospho-CREB1 immunoprecipated the DNA fragment containing the B fragment CRE element in cAMP-treated RAW264.7 cells but not in control cells. In agreement with our gel shift experiments, ChIP analysis confirms that phospho-CREB1 specifically binds the functional CRE found in the first intron of the murine ABCA1 gene in cAMP-treated RAW264.7 cells.
A STAT3/4 Site Nearby the b′ CRE Is Required for cAMP-Mediated Activation
To further characterize the mechanism by which the B fragment CRE can mediate the induction by cAMP, constructs containing a shortened b′ fragment were generated and analyzed via transfection in RAW264.7 cells. As shown in Figure 4A, the 366 bp b′ construct displayed a significant 55-fold cAMP induction of luciferase activity (P<0.01) as compared with the pABCA1 parent construct. However, this induction was no longer observed if the CRE site was mutated (b′-Mut), confirming the requirement of the CRE site for cAMP induction. Deletion of 234 bp from the 5′ end (b′-1) did not affect the stimulation by 8Br-cAMP, whereas a 5′ 246-bp deletion (b′-2) completely abolished this induction, indicating that the 12 bp region at the 5′ end of the b′-1 construct was necessary for cAMP induction. Thus, although the CRE site was absolutely required for the cAMP-mediated activation, it was not sufficient and a 12-bp sequence located 81 bp away was also required for cAMP induction.
Mutations in this 12-bp sequence (1 to 9; Figure 4B) indicated that mutations 1, 2, 4, and 5 led to a complete loss of induction of luciferase activity by 8Br-cAMP (b′-1-M1, b′-1-M2, b′-1-M4, and b′-1-M5 constructs; Figure 4A), whereas mutations 3 and 6 had either no (b′1-M3) or a weak effect (b′-1-M6). Additional mutations 7, 8, and 9 had no effect on the induction of the luciferase activity by cAMP (data not shown). Taken together, these results suggested that the core sequence element 5′-GGxAA-3′ in the 12-bp sequence was required for cAMP induction.
This core sequence element matched the consensus binding sites for STAT3 and STAT4 (5′-GGGAA-3′). Using a double-stranded 28-bp biotinylated oligonucleotide probe (STAT-b′-1) spanning this region, gel shift assays were performed to test whether STAT3 or STAT4 binds to this fragment. Incubation of the biotinylated probe with nuclear extracts of either control or 8Br-cAMP–treated RAW264.7 cells led to the formation of 2 DNA-protein complexes (I and II) (Figure 4C, lanes 2, 3 and 10), which were not observed in the presence of an excess of nonbiotinylated probe (lanes 4 and 11), whereas mutations in the biotinylated probe (M4 and M5), corresponding to mutations that neutralized cAMP induction (Figure 4A), prevented the formation of the 2 complexes (lanes 6 to 9). Competitions with an excess of nonbiotinylated probes for ISRE (lane 5), STAT1 (lane 12), or IRF-1 (lane 15) probes did not affect the formation of complexes I and II, whereas an excess of STAT3 (lane 13) and STAT4 (lane 14) probes abolished the formation of the complex I and II, respectively. Thus, the element 81 bp away from the B fragment CRE could bind both STAT3 and STAT4 in a cAMP-independent fashion.
Dominant-Negative CREB Inhibits cAMP-Mediated Induction of ABCA1 in RAW264.7 Cells
If the binding of CREB to the B fragment CRE is required for cAMP induction of ABCA1 expression in RAW264.7 cells, then we would expect that expression of a dominant-negative CREB (A-CREB21) would prevent the cAMP dependent enhancer activity of the B fragment and abolish the cAMP-mediated stimulation of ABCA1 expression. The cAMP mediated induction of ABCA1 was drastically reduced (−71%, P<0.01) with increasing amounts of A-CREB, demonstrating that expression of the dominant-negative CREB prevented the CREB-mediated activation through the B fragment in response to cAMP (Figure II, available online at http://atvb.ahajournals.org).
The involvement of CREB in the cAMP-mediated stimulation of ABCA1 expression was confirmed by Western blot after nucleofection of A-CREB in RAW264.7 cells (40% transfection efficiency). The level of ABCA1 protein was strongly induced in 8Br-cAMP–treated RAW264.7 cells after nucleofection with the control expression vector (Figure III, available online at http://atvb.ahajournals.org). However, nucleofection of the dominant-negative A-CREB expression vector led to a 49% reduction of the cAMP-mediated induction of ABCA1 (P<0.001), providing evidence that activated CREB is required for the cAMP-mediated induction of ABCA1 in RAW264.7 cells.
Protein Kinase A Mediates the cAMP Induction of ABCA1 in RAW264.7 Cells
Activation of protein kinase A (PKA) by cAMP is the general mechanism by which CREB is phosphorylated and activated. Cholesterol efflux to apoAI was strongly induced by 0.3 mmol/L 8Br-cAMP; however, addition of the PKA inhibitor H-89 reduced, in a dose-dependent fashion, ABCA1-mediated cholesterol efflux to apoAI and the levels of ABCA1 protein (Figure IV, available online at http://atvb.ahajournals.org). Thus, the activation of PKA is absolutely required for the induction of ABCA1 by cAMP.
Overall, our data support a model in which cAMP activates PKA leading to CREB1 activation that binds to a strong CRE in the first intron of the mouse ABCA1 gene and induces its transcription. The role of CREB1 in the cAMP-mediated induction of ABCA1 was confirmed by the use of a dominant-negative CREB that dimerizes with wild-type CREB and prevents binding to CRE.21 The dominant-negative CREB prevents the binding of CREB1 to a CRE without affecting the ability of others members of the ATF/CREB family to bind the CRE,21 suggesting that CREB1, and not other ATF factors, is required for the ABCA1 induction by cAMP.
We first used a bioinformatic approach, based on the finding consensus CRE sites in the mouse ABCA1 gene, to identify functional CREs, but in this case, this method was not efficient or successful. However, a shotgun cloning strategy was successful, and one of the reasons that this method was superior may have been that the strongest cAMP responsive enhancer required more than just a functional CRE consensus sequence. We determined that the strong CRE site in the first intron of the mouse ABCA1 gene could only mediate cAMP responsiveness of a reporter gene in transfection studies with the cooperation of a STAT element located 81 bp from the CRE site.
Gel mobility shift assays indicated that STAT3 and STAT4 could bind this STAT element, suggesting that STAT3 and/or STAT4 may interact with CREB1 on the enhancer in the first intron of the mouse ABCA1 gene. Recombinant CREB has previously been shown to bind to STAT1–3 in pull-down experiments,23 suggesting that STAT1–3 and CREB may form a transcriptional complex. Several studies also reported that the transcriptional coactivator CREB binding protein (CBP) can bind to various STATs.24–26 A recent study suggested that the association of phospho-CREB1 with a number of coactivators, such as CBP, is too weak for gene activation and that additional regulatory partners are needed for stable recruitment of such cofactors to the promoter.27 It is possible that the binding of STAT3 and/or STAT4 on the STAT element may help stabilize the recruitment of CBP by phospho-CREB1 on the CRE during cAMP induction of the murine ABCA1 gene. To our knowledge, this study is the first demonstration of the requirement for both a CREB and STAT3/4 element for the cAMP-mediated activation of gene expression.
Bortnick et al demonstrated that elevation of intracellular cAMP by prostaglandins E1 and E2 in mouse macrophages leads to an increase in ABCA1-mediated cholesterol efflux to apoAI, suggesting that G-protein–coupled prostanoid receptors could be involved in the stimulation of ABCA1 in this cell type.19 The multi-fold induction of ABCA1 gene expression by cAMP only occurs in mouse macrophages, whereas cAMP has little effect on ABCA1 expression in human cell lines.19,28 A weak induction (<2-fold) of both ABCA1 expression and cholesterol efflux to apoAI by cAMP has been observed in the human macrophage cell line THP-1.19,29 Furthermore, Cavelier et al reported that the human ABCA1 transgene is downregulated by a cAMP treatment whereas the endogenous murine ABCA1 gene is induced in peritoneal macrophages derived from transgenic mice expressing a human ABCA1 containing BAC.18
Both the mouse and human ABCA1 genes have 50 exons,20,30 and many large highly conserved noncoding elements have been identified.30 Although the sequence surrounding the functional CRE site in the first intron of mouse ABCA1 gene is 65% identical with the corresponding sequence in the first intron of the human gene, alignment of both sequences revealed that an 8-bp insertion disrupts the CRE site in the human sequence whereas the STAT element is conserved in both species (Figure V, available online at http://atvb.ahajournals.org). The absence of the functional CRE site in the first intron of the human ABCA1 gene could explain the lack of responsiveness of the human ABCA1 gene to cAMP. Because regulatory elements tend to be highly conserved among mammals, the nonconservation of the functional CRE site in the first intron of the human gene as well as the loss of a substantial induction of human ABCA1 gene expression by cAMP suggests that this pathway is not essential for regulation of ABCA1 in humans. However, despite of the lack of a role for cAMP in the induction of the human ABCA1 gene, it has been reported exogenous apoAI binding to cellularABCA1 increases intracellular cAMP levels, activates PKA, and induces phosphorylation and post translational activation of ABCA1 in human fibroblasts.31,32 Thus, although human and mouse ABCA1 do not share transcriptional activation by cAMP, they are both responsive to cAMP, albeit by different mechanisms.
This work was supported by grant RO1 HL66082 from the National Institutes of Health and a Pfizer International HDL Award.
- Received November 2, 2005.
- Accepted December 7, 2005.
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