Sterol upregulation of human CETP expression in vitro and in transgenic mice by an LXR element (original) (raw)
A reporter gene controlled by the human CETP proximal promoter is induced by sterol in differentiated 3T3-L1 adipocyte. Luciferase reporter genes controlled by the wild-type CETP promoter from 1 to –570 were transiently transfected into 3T3-L1 preadipocytes or differentiated adipocytes. The induction of the reporter gene by sterol was analyzed by incubating the cells in the absence or presence of 22(R)-hydroxycholesterol. The luciferase reporter gene activity was induced 2- to 3-fold by 22(R)-hydroxycholesterol in differentiated adipocytes. However there was no significant sterol induction in preadipocytes (Figure 1a). Similar but less consistent induction was also obtained by 25-hydroxycholesterol treatment in adipocytes (data not shown). The CETP promoter contains a sterol regulatory element (SRE), similar to that in the HMG CoA reductase promoter. However, mutation (Mut2) or deletion (ΔSRE) of the SRE in the CETP promoter did not abolish the sterol induction in adipocytes, consistent with the results in transgenic mice (28). These results suggest that differentiated 3T3-L1 adipocytes can be used as a model to analyze a SURE, distinct from the SRE, in the CETP promoter.
(a) Induction of the CETP promoter in adipocytes (shaded bar) but not preadipocytes (open bar). 3T3-L1 preadipocytes and differentiated adipocytes were transfected with reporter constructs in which the luciferase reporter gene is controlled by the proximal CETP promoter (from 1 to –570) with or without mutation in the SRE-like element (Mut2). The transfected cells were cultured in LPDS or LPDS + 4 μg/mL 22(R)-hydroxycholesterol for 24 hours. Luciferase activities were measured using the Promega Dual-Luciferase assay system, which uses Renilla luciferase as a control for transfection efficiency. The fold induction by sterol was calculated by dividing the luciferase activity in the presence of sterol by the activity in the presence of vehicle only. Means ± SD are shown. (b) RNA expression of LXRα and LXRβ in 3T3-L1 preadipocytes (lane 1) and differentiated adipocytes (lane 2). Northern blots were performed with equal amount of Poly A+ RNA isolated from 3T3-L1 preadipocytes and day 4 differentiated adipocytes using LXRα and LXRβ cDNA probe (21, 37).
Deletional analysis of the CETP promoter in 3T3-L1 adipocytes. To identify the region containing the SURE, luciferase reporter genes controlled by 5′ truncated CETP promoter elements were transiently transfected into 3T3-L1 adipocytes. Truncation of the region from –570 to –258 abolished the induction by 22(R)-hydroxycholesterol, suggesting the presence of a SURE in this region (Figure 2). To define further the region containing the SURE, a series of internal deletions in the CETP promoter were generated. Significant sterol upregulation was obtained in the construct containing deletion of the region from –370 to –258 as well as 5′ truncation to –430 (Figure 1a). The –370 to –430 region, linked to the –90 promoter, gave a positive sterol response, whereas deletion of the –370 to –413 sequence abolished sterol induction within the context of the longer –570 bp promoter construct (Figure 2). This result suggests that the region from –370 to –413 is responsible for the positive sterol response in the CETP promoter.
Deletional analysis of the CETP promoter in adipocytes. Reporter constructs containing various truncated proximal CETP promoter were transfected into 3T3-L1 adipocytes. The fold induction (means ± SD) by 22(R)-hydroxycholesterol is shown. *P < 0.005 versus –60-Luc; **P < 0.0005 versus –60-Luc).
The region from –370 to –430, which endowed a positive sterol response when linked to the CETP proximal promoter region, contains several sequences that could potentially bind transcription factors (Figure 3b). There is a 6-bp sequence, GGGTCA (–394 to –399), which conforms to the consensus nuclear hormone receptor binding element, PuGG/TTCA. This core sequence and the downstream CGGGCA sequence (although the latter is divergent from the nuclear receptor consensus element) could form a direct repeat separated by 4 nucleotides (DR4). DR4 elements have been shown to bind to nuclear receptors (30). Upstream of the putative DR4 element, there is a direct repeat of a sequence CAGGC separated by 5 nucleotides. To explore whether either of these elements is involved in the positive sterol response, these segments were mutated and the corresponding promoter constructs were evaluated in the luciferase assay (Figure 3a).
(a) Mutational analysis of CETP promoter activity in adipocytes and functionality of the CETP DR4 element in a heterologous TK promoter construct in adipocytes. Mutations were inserted into the CETP promoter with internal deletions (Δ90-370) or into the context of the long promoter (M2L–M5L). Mutations M1–M5 are shown in b. Reporter constructs in which reporter genes were controlled by the TK minimal promoter alone (TK-Luc), TK plus 3 copies of the CETP DR4 (3x-cpDR4-TK-Luc), or 3 copies of the 5′ half site of the DR4 element (3x1/2cpDR4-TK-Luc) were also used to analyze the function of the CETP DR4 element. Fold induction (mean ± SD) is shown. The results are from 3–5 independent experiments. *P < 0.0005 versus mutant constructs; #P < 0.005 versus M1-Luc. (b) Sequence of CETP promoter from –380 to –426. Mutations in this region are shown in italics. The putative DR4 element is shown in bold. M1 is identical to M1* except that there is an additional single nucleotide mutation in the DR4 element.
Mutations M1–M4 (Figure 3b) were introduced into short promoter reporter constructs (M1-Luc, M3-Luc, and M4-Luc) or were evaluated in the context of the full 570-bp promoter (M2L-Luc, M3L-Luc, and M5L-Luc). The M1 mutation, which disrupted the upstream CAGGC repeats and also has a single nucleotide mutation in the 5′ repeat of the DR4 element, and the M2 mutation, which changed the 5′ flanking sequence of the DR4, abolished the positive sterol response (Figure 3a). However, the M1* mutant, which abolished the upstream repeat without changing the DR4 element, had a normal response (Figure 3a). These results implicate the DR4 element. Other mutations that disrupted the DR4 repeats (M3 and M5) or spacing region (M4) also abolished the positive sterol response. DR4 mutations gave similar results in either the deleted short promoter or the full-length 570-bp promoter. These results suggest that the DR4 element might be the SURE. Consistent with this, a reporter gene controlled by 2 copies of the region containing the SURE (2×SURE-Luc) showed approximately 4- to 5-fold induction by sterol, a more robust response than the construct containing 1 copy of the SURE (Figure 3a).
Functionality of the SURE (CETP DR4) in a heterologous TK promoter construct. To confirm further the sterol upregulatory function of the DR4 element, DNA fragments containing 3 copies of the CETP DR4 (cpDR4) or the 5′ half site (GGGTCAttgtc) of the DR4 were cloned into a heterologous (TK) promoter construct. Insertion of 3 copies of the CETP DR4 upstream of TK led to a significant 7-fold sterol induction (Figure 3a, bottom). However, the reporter gene controlled by TK alone or the half element of DR4 was not induced by sterols. This result indicates that the CETP DR4 is sufficient for sterol upregulation and functions as a SURE in a heterologous promoter system.
Evaluation of the SURE in transgenic mice. To determine whether the CETP SURE identified in adipocytes also functions in vivo, we made CETP transgenic mice containing mutations in this element (Figure 4). In control NFR-CETP Tg mice, the high-cholesterol diet caused a significant 2.1-fold induction of plasma CETP activity. Deletion of the –413 to –370 region abolished this response, as shown in 5 different founders and in F1 mice from 1 founder. Transgenic founders bearing mutations in the upstream direct repeat within this region (M1*, Figure 3b) showed a normal response to the high-cholesterol diet, whereas founders with mutations in the first repeat of the DR4 element (M3, Figure 3b) had no response to the diet. These findings parallel the in vitro results and show that the DR4 element is involved in the response of the human CETP gene in vivo.
Effect of high-fat, high-cholesterol (HFHC) diet on the plasma CETP activity of mice expressing CETP transgenes controlled by wild-type (NFR-CETP), or mutated CETP promoters (MUT3-CETP, M1*-CETP, M3-CETP). Five different MUT3-CETP founders, 4 MUT3-CETP F1 mice, 2 M1*-CETP founders, 2 M3-CETP founders, and 3 control NFR-CETP mice were used. CETP activities (mass) were determined by an isotopic assay using plasma collected before or after an HFHC diet for 7 days. Fold induction (mean ± SD) of plasma CETP activity by the diet is shown. *P < 0.0005 versus NFR-CETP.
Specific protein-DNA interaction involving the CETP DR4 element. DNA mobility shift experiments were carried out to analyze transcription factors binding to the region containing the SURE. A 24-bp DNA fragment (cpDR4), corresponding to the putative DR4 element and its flanking sequence (–380 to –403) in the CETP promoter, was incubated with nuclear extracts from 3T3-L1 adipocytes. Excess cold F2 fragments, which consists of only the 5′ half site of DR4, was also included to compete away the binding of proteins to the DR4 half site (data not shown). A major band (P1) (Figure 5a, lane 1), representing protein-DNA complexes, was abolished by addition of excess unlabeled cpDR4 (Figure 5a, lane 2). Mutations in the 2 repeats of the DR4 elements (M3, M4, M5, and M1) abolished or reduced the binding of P1, indicating that P1 represents proteins binding to the DR4 (Figure 5a, lanes 4–7). The sequence flanking the DR4 also seems important for the binding of P1, as M2, which changed the upstream flanking sequence, competed poorly for the P1 binding (Figure 5a, lane 3). A short fragment, F6, which represents a truncated DR4, containing the 5′ repeat and spacing region, was unable to compete for P1 binding (Figure 5a, lane 8). These results suggest that P1 protein complex binds to CETP DR4 element and not to the half site of this element. The mutations (M1–M4) that decreased the binding of DNA to P1 protein complex also abolished the sterol upregulation in reporter assays (Figure 3a), indicating that the P1 protein complex probably contains the transcription factors that mediate the sterol upregulation.
(a) Gel mobility shift analysis to identify the proteins that bind to the DR4 element in the CETP promoter. A 24-bp fragment containing the CETP DR4 (cpDR4) (–380 to –403) was labeled and incubated with nuclear extracts from differentiated 3T3-L1 adipocytes in the presence of excess F2 fragment (CETP DR4 half site) to abolish nonspecific factors binding to the DR4 half site. Various cold competitor fragments were included as indicated, and shifted bands were resolved in a 5% polyacrylamide gel. F6 consists of only the 5′ half site and spacing region of the DR4 element. M1–M5 mutations are shown in Figure 3b. The arrow indicates the P1 complex. (b) The protein complex binding to the CETP DR4 contains an LXR/RXR heterodimer. Gel shift experiments were carried out in the absence (lane 1) or presence of 2 μg polyclonal antibodies against LXRα/β, LXRα, or RXRα (lanes 2–4), or RORα (lane 5). The arrow indicates the P1 band. The bands supershifted by RXRα or LXR antibodies are indicated by thick arrows.
Characterization of the transcription factors that bind to the CETP SURE. Two nuclear receptors, LXRα and LXRβ, are known to be activated by certain hydroxysterols and to bind DR4 elements in heterodimeric complexes with RXRα (31). To determine whether the P1 protein complex that binds to the CETP DR4 contains LXR and RXRα, we used antibodies against LXR or RXR in gel shift assays (Figure 5b). Polyclonal antibodies against the COOH-terminal 19 amino acids (424–442) of LXRα/β reduced the intensity of the P1 band (Figure 5b, lane 2) and also gave a weak supershifted band, suggesting the P1 complex contains LXRα and/or LXRβ(22). Another antibody (P20), which is against the 20 amino acids (370–389) near the COOH-terminus of LXRα, blocked the binding of the proteins to DNA (Figure 5b, lane 3). Two supershifted bands were observed in the presence of RXRα antibody (Figure 5b, lane 4), suggesting that the protein complex also contains RXRα. It is not known why RXRα antibody typically produces 2 supershifted bands. A control antibody (against RORα) had no effect on the P1 complex (Figure 5b, lane 5). Furthermore, The P1 band was competed away by the Cyp7a LXRE, which is known to bind LXRα (19) (data not shown). These data indicate that the P1 complex contains LXRα and RXRα. However, these results do not exclude the possibility that LXRβ is also present in this complex.
LXRα mRNA was found to be markedly induced in differentiated 3T3-L1 adipocyte cells (Figure 1b). The induction of LXRα during adipocyte differentiation is correlated with the appearance of sterol induction of the CETP promoter in differentiated adipocytes (Figure 1a). However, LXRβ mRNA expression showed little change during differentiation (Figure 1b).
Effect of 9-cis retinoic acid on CETP promoter activity. Gel shift assay data suggest that LXR/RXRα binds to the CETP DR4. It has been shown that RXRα in the LXR/RXR complex plays an active role, in that RXRα/LXR heterodimers can be activated by either RXR ligands (i.e., 9-cis retinoic acid), LXR ligands [i.e., 22-(R)-hydroxycholesterol], or additively by both LXR and RXRα ligand (21, 23, 31). To find out whether RXRα is involved in the function of the CETP SURE, we analyzed the effect of 9-cis retinoic acid on the CETP promoter-reporter activity (Figure 6a). The expression of the reporter luciferase controlled by the CETP promoter (Δ90-370-Luc) was induced approximately 2-fold in the presence of 9-cis retinoic acid. An additive induction was observed in the presence of both 22(R)-hydroxycholesterol and 9-cis retinoic acid. Mutation of the DR4 element (M3–M5) and the 5′ flanking sequence (M2) abolished both the retinoic acid and sterol induction. Furthermore, the expression of a reporter gene controlled by 3 copies of the DR4 in a heterologous TK promoter (3xcpDR4-TK-Luc) was highly induced by 9-cis retinoic acid and 22(R)-hydroxycholesterol, and an additive effect (∼23-fold) was also obtained (Figure 6b). These results show that RXRα is functionally involved in the upregulation of the CETP promoter and that it acts on the DR4 element.
Additive effects of sterols and 9-cis retinoic acid on CETP promoter activity, mediated via the SURE. Reporter constructs that contain the wild-type CETP promoter (Δ90-370-Luc) or promoters with mutations in the DR4 element (M3-Luc) (a) or 3 copies of the CETP DR4 in a heterologous TK promoter (3xcpDR4-TK-Luc) (b) were transfected into differentiated 3T3-L1 adipocytes. Transfected cells were then cultured in LPDS medium in the presence of 4 μg/mL 22(R)-hydroxycholesterol, 1 μM 9-cis retinoic acid or both. The fold induction (mean ± SD) from 3 independent experiments is shown except for M2L-luc and M4-Luc, which are in duplicate, and 3xcpDR4-TK-Luc, which is in triplicate.
Transactivation of CETP promoter by LXRα and LXRβ. To evaluate further the role of LXRs in sterol activation of the CETP promoter, we analyzed the ability of LXRα and LXRβ to transactivate the CETP promoter by cotransfecting the CETP promoter-reporter with LXRα/RXRα or LXRβ/RXRα in CV-1 cells (Figure 7). Transfection of LXRα/RXRα or LXRβ/RXRα increased the basal expression of the luciferase reporter controlled by the CETP promoter (Δ90-370-luc) by approximately 2- or 5-fold, respectively. With sterols, the induction significantly increased to approximately 7- to 10-fold in cells expressing LXRα/RXRα or LXRβ/RXRα. Mutation of the DR4 element (M3) abolished transactivation by LXRs. LXRs also markedly transactivated the promoter containing 3 copies of the CETP DR4 (SURE). Expression of LXRα/RXRα or LXRβ/RXRα increased the basal activity of 3xcpDR4-TK promoter by approximately 3- to 5-fold and dramatically induced expression (17- to 18-fold) in the presence of sterols. These results indicate that LXRα or LXRβ can mediate sterol induction of the CETP gene via the DR4 element.
Transactivation of the CETP promoter or a synthetic promoter containing 3 copies of the DR4 from the CETP promoter (3xcpDR4) by LXRα and LXRβ in CV-1 cells. CV-1 cells were transfected with Δ90-370-Luc, M3-luc, or 3XcpDR4-TK-Luc with or without cotransfection with LXRα/RXRα or LXRβ/RXRα. The transfected cells were then cultured in LPDS ± 4 μg/mL 22 (R)-hydroxycholesterol. Three experiments were carried out in duplicate for Δ90-370-Luc and 3XcpDR4-TK-Luc. Two experiments were carried out for M3-Luc. #P < 0.001 versus the same condition without cotransfection with LXRα/RXRα or LXRβ/RXRα. *P < 0.05 versus the same conditions in the absence of sterols.