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Identification of beta-carotene 15, 15'-monooxygenase as a peroxisome proliferator-activated receptor target gene

ANA BOULANGER^*, PAMELA MCLEMORE^*, NEAL G. COPELAND, DEBRA J. GILBERT, NANCY A. JENKINS, SHIRLEY S. YU^*, SUSAN GENTLEMAN^* and T. MICHAEL REDMOND^*,2

^* Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA; and
Mouse Cancer Genetics Program, National Cancer Institute at Frederick, Frederick, Maryland, USA

²Correspondence: NEI-LRCMB, NIH, Bldg. 6, Rm. 339, 6 CENTER DR MSC 2740, Bethesda, MD 20892-2740, USA. E-mail: redmond{at}helix.nih.gov

SPECIFIC AIMS

We wished to characterize the promoter of the mouse gene (Bcm) for ß-carotene 15,15'-monooxygenase (BCM), which catalyzes the first step of vitamin A biosynthesis from provitamin A carotenoids, and determine the factors underlying the transcriptional regulation of this gene.

PRINCIPAL FINDINGS

1. Cloning of the Mouse Bcm gene and identification of putative transcription factor binding sites
The Bcm gene, including its 5'- and 3'-flanking regions, spans 40 kb. By alignment of the cDNA and the genomic sequences, intron-exon boundaries in the Bcm gene were defined, giving 11 exons and 10 introns in the mouse Bcm gene (data not shown). The mouse chromosomal locus (given the name Bcdo) for Bcm is on distal chromosome 8. Analysis of the 2 kb 5'-flanking region showed several putative transcription factor binding sites, including a TATA and a CACA box at positions –28 to –22 and –41 to –32, respectively. Upstream of these boxes there is a direct repeat of the DR1 type, (TGACCTTTGACCT), a potential peroxisome proliferator response element (PPRE) recognition site for the peroxisome proliferator-activated receptor (PPAR). This is followed by three consensus E-box sites, a Hox 1.3 bicoid site, and a contiguous bHLH-AP2 sequence at positions –96 to –85 and –72 to –61, respectively.

2. The PPRE site is involved in the transcriptional activation of Bcm
The 5'-flanking region DNA fragment of 2 kb was cloned into the luciferase reporter vector pGL3-Basic and transfected into four cell lines: TC7, PF11 (both expressing high levels of BCM mRNA), monkey RPE primary culture (low level expression of BCM mRNA), and ARPE-19 (no expression of BCM mRNA). TC7 and PF11, derived from the human intestinal cell line Caco-2, are the only two cell lines known to date to possess high BCM enzyme activity in vitro. TC7 and monkey RPE cells gave a 60- and 30-fold increase, respectively, in reporter activity compared with pGL3-Basic (Fig. 1 A); high activation was seen in PF11 cells (data not shown) but low activity was seen in ARPE-19 (6-fold increase). To map the regulatory elements, we made four Bcm-luciferase nested constructs based on the position of the different predicted elements in the Bcm promoter sequence. The shortest clone, S7, lacked any putative transcription element; S4 added the TATA and CACA elements, a PPRE site, and a putative bHLH/AP2 sequence; S3 added the upstream putative Hox 1.3 bicoid site; and S2 added the upstream AP2 site. Comparison of these (Fig. 1B ) showed that S4 contained the essential region for inducing Bcm promoter activity in TC7 and monkey RPE cell lines. No significant difference (<2-fold) was made by the addition to S4 of upstream sequences. Much lower promoter activity was observed when the S7 fragment construct was transfected into any of the three cell lines. Thus, there is a sharp rise in the promoter activity between the regions represented by plasmids pS4luc and pS7luc. Next we made two additional constructs, one containing the TATA and CACA boxes alone (S6) and the other containing these plus the upstream PPAR binding site (S5). Figure 1C shows that S5 drives luciferase activity indicating the presence of a positive element within this region. Since basal activity is maintained with the S6 fragment (pS6luc plasmid, sequence from –41 to +163) and the promoter is similarly active in the three cell lines, we can consider it the minimal region necessary to drive the basal activity (core promoter). Addition of the PPRE increases basal promoter activity by four- and twofold in TC7 and monkey RPE cells, respectively. To confirm the involvement of the PPRE in the Bcm promoter response, we abolished it by site-directed mutagenesis, decreasing the activity of the mutated plasmid (PS2mluc) by threefold compared with PS2luc (Fig. 1D ).

View larger version (26K): [in this window] [in a new window]   Figure 1. A PPRE element in the Bcm promoter is required for activity. Different Bcm 5'-flanking fragments were cloned into a promoter-less vector (pGL3-Basic) with luciferase as a reporter gene and transfected into TC7, ARPE-19 cell lines and monkey RPE primary cultured cells. A) A 2.2 kb promoter fragment (S1; –2031 to +163) was used to determine the functionality of the Bcm promoter. The pGL3-Control vector (luciferase gene driven by SV40 promoter) was used as a positive control to monitor the entire procedure. B) Serial deletions were made by PCR at the 5'-end of the Bcm promoter (identified by name (length: transcriptional elements contained)): S2 (–289 to +163: TATA, CACA, PPRE, bHLH/AP2, Hox1.3 bicoid, and upstream AP2 elements), S3 (–107 to +163: TATA, CACA, PPRE, bHLH/AP2, and Hox1.3 bicoid elements), S4 (–76 to +163: TATA, CACA, PPRE, bHLH/AP2 elements), and S7 (–18 to +163: no elements) to investigate the regulatory sequences of the Bcm promoter. C) Identification of an enhancer and a core promoter region. Transient transfections of DNA fragments between –60 and +163 (S5: TATA, CACA, and PPRE) and –41 and +163 (S6: TATA and CACA). D) Effect of mutations in the PPRE site. TC7 cells were transfected with wild-type plasmid pS2luc or mutated plasmid pS2mluc. The activity of the luciferase reporter gene was expressed as fold relative to the activity of pGL3-Basic (which was assigned an activity value of 1.0) in the 4 sets of experiments (A–D). The means and SE are shown (n>3).

3. PPAR interacts with the PPRE site
To determine whether cell lines expressing BCM contain transcription factors capable of binding to the predicted PPRE site in the Bcm promoter, we performed EMSA using TC7, PF11, and monkey RPE nuclear extracts with synthetic oligonucleotides (sense and antisense) corresponding to the region containing these sites. TC7 and PF11 nuclear extracts gave similar shift mobility, but a higher relative shift mobility was seen with the monkey RPE nuclear extract (comparison not shown). To identify the proteins involved, antibodies to PPAR and PPAR or the corresponding anti-rabbit preimmune serum were added to the complexes in a supershift assay. Both anti-PPAR antibodies, but not preimmune serum, supershifted the DNA–protein complex (data not shown), indicating that PPAR (most likely PPAR) specifically binds to this site.

4. The Bcm promoter responds to peroxisome proliferators
PPAR typically binds the PPRE site as a heterodimer with the retinoic X receptor (RXR). Thus, to characterize the functionality of the PPRE site, we used the specific PPAR agonists Ciglitazone and LY 17883 and the RXR agonist 9-cis-retinoic acid (9cRA) in our transient transfection assay with the S5 construct, either alone or combining each PPAR agonist with 9cRA. Cotransfection with RXR and PPAR expression vectors was used to confirm the responsiveness of the Bcm PPRE element, though we found both to be present in TC7 cells. RXR and PPAR mRNA was shown to be present in TC7 cells by semiquantitative RT-PCR. Cotransfection of TC7 cells with S5 and a pSG5-PPAR expression vector showed a significant twofold increase in the presence of 20 µM Ciglitazone that was augmented to almost threefold when Ciglitazone was added along with 9cRA (Fig. 2 A). Cotransfection of TC7 cells with S5, along with the pSG5-RXR expression vector, and treatment with 9cRA alone gave a fivefold increase in luciferase activity over control (cotransfected cells with DMSO vehicle). This typical response, documented in other systems, was due to 9cRA-induced RXR homodimerization. Next, S5 cotransfection experiments were conducted with PPAR and RXR expression vectors. An almost threefold increase was obtained when TC7 cells were treated with Ciglitazone without 9cRA. The increase in reporter gene activity in the presence of 9cRA alone was not significant in this case (Fig. 2A ). Thus, luciferase activity obtained by cotransfecting PPAR and RXR was due to the PPAR ligands acting via the heterodimer PPAR/RXR and not to the 9cRA acting via RXR homodimers. Moreover, luciferase activity was significantly increased to five- to sixfold when each PPAR agonist was added in combination with 9cRA, indicating that Ciglitazone and PPAR-mediated activation was synergistically increased by 9cRA. Deletion of the PPRE site (construct S6) rendered the promoter unresponsive to the PPAR agonists used (Fig. 2B ). To test in vivo regulation of expression of BCM by PPAR, mice were treated with WY 14643, an agonist with activity toward PPAR and PPAR. Mice treated with this agonist showed a significant increase in the expression of BCM associated with the detergent soluble fraction and to a lesser degree with the cytosolic fraction of liver homogenates (data not shown).

View larger version (37K): [in this window] [in a new window]   Figure 2. The PPRE element in the Bcm promoter responds to PPAR and RXR agonists. TC7 cells were transfected using 2 µg of the reporter plasmid S5 (A) or S6 (B) either alone (S5 (or S6)) or in combination with 2 µg of expression vectors PPAR (S5 (or S6)/PPAR), RXR (S5 (or S6)/RXR), or both (S5 (or S6)/PPAR/RXR) in the presence of different pharmacologic compounds added in the culture medium either alone or in combination: Ci, 20 µM Ciglitazone; RA, 2 µM 9-cis-retinoic acid; Ci/RA, 20 µM Ciglitazone and 2 µM 9-cis-retinoic acid. The activity of the luciferase reporter gene was expressed as fold relative to the activity obtained by the vehicle DMSO depending on the transfection performed (which was assigned an activity value of 1.0) in the 2 sets of experiments (A, B). The means and SE are shown (n>3).

CONCLUSIONS AND SIGNIFICANCE

BCM is the first enzyme in the pathway for the biosynthesis of vitamin A from ß-carotene by animals. Here we demonstrate that a PPRE site located in the proximal region of the mouse Bcm promoter allows PPAR activators Ciglitazone and LY 17883 to regulate the expression of the Bcm gene via the heterodimerization of PPAR with the RXR receptor (Fig. 3 , top). However, 9-cis retinoic acid activated-RXR can regulate Bcm gene expression when PPAR ligand is absent (Fig. 3 , bottom). Significantly, deletion or mutation of the PPRE site causes promoter activity to revert to basal promoter activity. Consequently, we conclude that PPRE is the promoter element responsible for the restricted expression of Bcm. These data suggest that PPAR and its ligands may play a significant role in the biosynthesis of vitamin A, providing a broader function for PPARs in the regulation of carotenoid metabolism. The cellular retinol binding protein II (CRBPII) gene, preferentially expressed in adults in the small intestine, is the only other gene involved in carotenoid or retinoid metabolism known to contain PPRE sites. This gene is also up-regulated by fatty acids probably via PPAR and can be retinoic acid activated via RXR. BCM and CRBPII act successively in the metabolism of ß-carotene. The former cleaves ß-carotene and the latter binds the all-trans-retinal product, allowing for its reduction to retinol by retinal reductase and the subsequent esterification by lecithin:retinol-acyltransferase (LRAT) to retinyl esters for export from the enterocytes. Since CRBPII and Bcm (our results) are both regulated by PPAR ligands, PPAR may in turn sequentially up-regulate the expression of both proteins to metabolize ß-carotene. These data strongly suggest that dietary components (e.g., fatty acids, retinoids) affect the regulation of the expression of the Bcm gene and thus expand the role of PPARs in carotenoid and retinoid metabolism, consistent with their established role in neutral lipid metabolism and transport. Further exploration of how PPAR ligands and in vivo physiological stimuli affect Bcm transcription in the mouse is warranted.

View larger version (16K): [in this window] [in a new window]   Figure 3. Schematic diagram of the ligand-dependent Bcm transcriptional regulation via a PPRE site. PPAR specific agonists induce PPAR/RXR heterodimerization, which in turn activates Bcm gene expression. RXR-specific agonists alone can also activate Bcm via RXRa homodimerization. The PPRE site contained in the Bcm promoter is responsible for this ligand-dependent regulation and deletion of this site leads to an inhibition of transcription.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0690fje; doi: 10.1096/fj.02-0690fje

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