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β-Carotene regulates expression of β-carotene 15,15′-monoxygenase in human alveolar epithelial cells
Archives of Biochemistry and Biophysics 539 (2013) 230–238
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b-Carotene regulates expression of b-carotene 15,150 -monoxygenase in human alveolar epithelial cells Xiaoming Gong a, Raju Marisiddaiah b, Lewis P. Rubin a,⇑ a b
Department of Pediatrics, Texas Tech University Health Science Center, Paul L. Foster School of Medicine, El Paso, TX 79905, USA Children’s Research Institute, St. Petersburg, FL 33701, USA
a r t i c l e
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Article history: Available online 23 September 2013 Keywords: b-Carotene 15,150 -monooxygenase Retinaldehyde Retinoic acid PPARc RXRa Transcriptional regulation Alveolar epithelium
a b s t r a c t b-Carotene 15,150 -monooxygenase (CMO1, BCMO1) converts b-carotene to retinaldehyde (retinal) and is a key enzyme in vitamin A metabolism. CMO1 activity is robust in the intestine and liver, where cmo1 gene transcription may be subject to negative feedback by accumulation of its metabolic products. Evidence from CMO1 null animals also indicates that non-gastrointestinal CMO1 may be required for tissue-specific conversion of b-carotene into vitamin A. The aim of this study was to investigate the effects of the enzymatic substrate, b-carotene, on regulation of CMO1 in a cell model of human alveolar pneumocytes. We demonstrate that CMO1 is expressed in human alveolar epithelial (A549) cells and converts b-carotene into retinal and biologically active retinoic acids (RA). Exposure to b-carotene suppresses CMO1 expression at both mRNA and protein levels. b-Carotene, but not all-trans RA, decreases CMO1 promoter activity in a time- and dosage-dependent manner. This b-carotene-mediated inhibition of CMO1 expression results from decreased binding of peroxisome proliferator-activated receptor c (PPARc) and retinoid X receptor a (RXRa) in the CMO1 promoter. b-Carotene treatment also antagonizes PPARc activity in HEK293 cells that stably express CMO1 wild-type, but not in cells that express the CMO1 mutant or vector alone. These findings have implications for local vitamin A synthesis in the lung, especially during systemic vitamin A insufficiency and may also help to explain, in part, the mechanism underlying the increased lung cancer risk upon b-carotene supplementation in smokers. Ó 2013 Elsevier Inc. All rights reserved.
Introduction Vitamin A (retinol) is a fat-soluble micronutrient essential for normal morphogenesis and epithelial differentiation and maintenance [1,2]. The importance of vitamin A throughout the life cycle is well established [3] and begins in embryogenesis [3,4]. Vitamin A exerts its biological actions largely through its principal active metabolites, specific retinoic acid (RA) isomers. RA activity, in turn, is mediated by binding to RA receptors (RARa, RARb, RARc) and retinoid X receptors (RXRa, RXRb, RXRc), ligand-inducible transcription factors which are members of the superfamily of nuclear hormone receptors [5]. RARs and RXRs form ligand-dependent heterodimers that bind to regulatory regions in specific target genes and modulate gene transcription [6]. Circulating vitamin A concentrations must be maintained in narrow range in order to avoid either deficiency or toxicity. Consequently, vitamin A production is tightly controlled for normal physiological processes.
⇑ Corresponding author. Address: Department of Pediatrics, Texas Tech University Health Science Center, Paul L. Foster School of Medicine, 4800 Alberta Avenue, El Paso, TX 79905, USA. Fax: +1 915 545 0918. E-mail address: [email protected] (L.P. Rubin). 0003-9861/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2013.09.013
A major dietary source of vitamin A is the pro-vitamin A carotenoids, particularly b-carotene, which is ingested from many vegetables and fruits. In humans, increased b-carotene intake leads to increased plasma concentrations of b-carotene and its metabolites. b,b-carotene 15,150 -monooxygenase (1CMO1, BCMO1) symmetrically cleaves b-carotene to yield two molecules of all-trans retinal (retinaldehyde) [7–10]. Studies undertaken in CMO1 null mice and in human subjects further establish the fundamental role of this enzyme in producing vitamin A aldehyde (retinaldehyde, or retinal) from dietary b-carotene [11,12]. Retinal is converted to retinol, the transport and storage form of vitamin A, and is further oxidized to RA. CMO1 expression and enzyme activity are robust in digestive sites in intestinal mucosa and liver [13] but the enzyme also is expressed in peripheral tissues [14,15]. In humans, unlike rodents, substantial amounts of absorbed b-carotene are not enzymatically cleaved in the intestine [16] and up to 15–30% of absorbed b-carotene remains intact and is delivered to peripheral tissues [17,18].
1 Abbreviations used: CMO1, b-carotene 15,150 -monooxygenase; ATRA, all-trans retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR-response element; EMSA, electrophoretic mobility shift assay.
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Local tissue-specific b-carotene to retinal metabolism may contribute to vitamin A production in extra-intestinal tissues. b-Carotene metabolism and CMO1 activity are present in human and rodent lung tissue [19–21]. However, the molecular details of how b-carotene metabolism in the lung is regulated are largely unknown. Although CMO1 enzymatic biochemistry has been studied for several decades, regulation of CMO1 gene expression and enzyme activity has largely focused on the intestinal tract, where CMO1 is subject to transcriptional feedback inhibition by RA [22]. Intestinal cell CMO1 gene expression in rodents also is stimulated by PPARc agonist and PPARc/RXRa heterodimer binding to a PPAR regulatory element (PPRE) [23]. In human intestinal cells, PPARc is essential but not sufficient to activate human CMO1 gene expression which is instead dependent on cooperation between PPARc and MEF2 isoforms [24]. More recently, an intestine specific homeodomain transcription factor, ISX, has been found to regulate CMO1 and scavenger receptor class B type 1 (SR-BI) expression, also via metabolic feedback [25,26]. Mechanisms of CMO1 regulation in extra-intestinal sites remains less studied. In the present report, we examined the effects of b-carotene on the expression of CMO1 in pulmonary alveolar epithelial cells. Lung terminal air sacs, or alveoli, are an important target for retinoid action, especially in development and during repair. We present evidence that b-carotene, but not ATRA, decreases CMO1 expression and inhibits CMO1 promoter activity through suppression of PPARc/RXRa binding to the CMO1 promoter. Moreover, a CMO1-dependent decrease in PPARc activity by b-carotene appears to be a key determinant in regulation of CMO1 expression. Materials and methods Plasmids and chemicals The plasmid pGL3-basic (Promega, Madison WI) and constructs for wild type, truncated and mutated pGL3-BCO1-Luciferase (pGL3-BCO1-Luc) reporter genes, pPPRE-tk-Luc reporter gene, pRARE-200Luc reporter gene, the expression vectors for PPARa, b, and c, RARb, RXRa, pCMV-b-Gal and pcDNA3.1 were described previously [24]. The pCMV-BCO1 expression vector was the gift of Dr. Stefan Andersson (Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, Texas). Dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), b-carotene, retinaldehyde and all-trans retinoic acid (ATRA) were from Sigma–Aldrich (St. Louis, MO). The PPARc agonist, GW1929, was obtained from Alexis Biochemicals (San Diego, CA). Antibodies The synthetic peptide [Ac-C]TKKQAASEEQRDRASD-Amide, corresponding to the C-terminal 15 amino acid residues 525–540 in human CMO1, was coupled to keyhole limpet hemocyanin and used for immunization of rabbits as described previously [27]. Polyclonal antibodies to CMO1 were characterized by an enzyme-linked immunosorbent assay using the synthetic peptide as antigen and by Western blot analysis.
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fetal bovine serum, were purchased from Invitrogen (Carlsbad, CA). Equal numbers of A549 cells (1 106/well) were plated into six-well cell culture plates for 24 h before addition of various treatments. b-carotene (stock solution, 1 mM) was dissolved in DMSO:THF (4:1). ATRA and retinaldehyde (in DMSO) at a concentration of 1 mM were prepared freshly before each experiment. Control cells were incubated with vehicle alone. Site-directed mutagenesis A panel of mutant CMO1 was made using a pcDNA3-CMO1 construct as template. The 5 residues of human CMO1 under study (His172, His237, His308, Glu450, His514) as described previously [28] were mutated to alanine using QuikChange™ multiple site-directed mutagenesis kit (Stratagene). The validity of all point mutations and integrity of the open reading frame were verified by DNA sequencing. Stable expression of wild-type and mutated CMO1 in HEK293 cells Stable CMO1 wild type (CMO1-wt), mutated (CMO1-mt) and pcDNA3.1 cell lines were established as previously described [29]. Briefly, the pcDNA3.1 vector and pcDNA3.1 plasmids containing the full-length wild type or [H172, H308, E450, H514] CMO1 mutants were transfected into HEK293 cells using Lipofectamin 2000 reagent (Invitrogen) and cells were selected in the presence of geneticin (G418, Gibco-BRL) (400 lg/ml). Isolated clones were selected by limited dilution and then expanded. Stable transfected clones were selected in the presence of G418 (500 lg/ml) and verified with PCR using primers for CMO1 and western blot using CMO1 antibody. Extraction of carotenoids and retinoids b-Carotene and its metabolites and retinoids were extracted as described previously, with minor modifications [30]. After incubation of cells with known amounts of b-carotene for the indicated times, culture plates were placed on ice, medium removed and monolayers washed with 0.5 ml of 10 mM sodium taurocholate in phosphate-buffered saline (PBS) to remove surface-bound carotenoids. After PBS washes 2, cells were harvested by brief trypsinization, cell pellets were homogenized in 0.5 ml ice-cold PBS and transferred it to glass test tubes. An aliquot (0.1 ml from 0.2 mM stock) of butylated hydroxytoluene (BHT) was added to the homogenate and, when required, a known amount of internal standard (echinenone) was added. Homogenates were extracted with 1.5 ml dichloromethane/methanol (1:2, v/v) and hexane. Following centrifugation, the resulting upper hexane-dichloromethane layer was collected. The lower layer was extracted two more times and the hexane-dichloromethane layer was combined with the initial extract. The combined extract was dried, re-dissolved in 0.1 mL dichloromethane/methanol (1:4, v/v) and subjected to HPLC analysis. We also analyzed the concentration of b-carotene in the medium before and after incubations. Sample handling, homogenization and extraction were carried out under cold conditions and dim yellow light to minimize carotenoid isomerization and oxidation.
Cell culture and treatments HPLC analysis of b-carotene and retinoids Human pulmonary alveolar epithelial A549 cells and HEK293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with penicillin (100 units/ ml), streptomycin (100 lg/ml), and 10% fetal bovine serum (HyClone, Logan, UT) at 37 °C in a humidified atmosphere of 95% air/ 5% CO2. All the above-mentioned reagents, with the exception of
b-Carotene and retinoids were analyzed and quantified as described previously, with minor modifications [31]. A Shimadzu HPLC system (Model: UFLC, Kyoto, Japan) equipped with PDA detector, SPD-M20A monitoring from 210 to 670 nm, comprising of a gradient pump system, LC-20AT and a personal computer equipped with LC Solution (Shimadzu) software was used for the
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detection and quantification of b-carotene and retinoids. b-Carotene and carotenoid metabolites were separated on a C30 carotenoid column (5 lm, 4.6 150 mm, YMC; Waters), attached to a guard cartridge (5 lm, 4.0 20 mm, YMC; Waters). Isocratic analysis was performed at a flow rate of 1 ml/min using acetonitrile:methanol:dichloromethane (60:30:10, v/v/v) containing 0.1% ammonium acetate (mobile phase A) using detection at 450 nm for b-carotene and 325, 350 and 376 nm, respectively, for retinol, retinoic acid and retinal. When necessary, the b-carotene cleavage product, retinal, was separated on a C18 column (5 lm, 4.6 150 mm, Biobasic-18; Thermo Scientific) attached to a guard cartridge (5 lm, 4 10 mm, Biobasic-18; Thermo Scientific) using acetonitrile:water (90:10, v/v) containing 0.1% ammonium acetate (mobile phase B) at a flow rate of 1 ml/min isocratically. The individual peak identities were confirmed by their characteristic absorption spectra using a PDA detector and were quantified by individual peak AUC referenced to the respective standard. RNA preparation and semi-quantitative RT-PCR Total RNA was isolated from control and treated A549 cells with TRIzol reagent (Invitrogen) and purified using the RNeasy system (Qiagen). About 1 lg of total RNA was reverse-transcribed using iScript™ cDNA Synthesis (Bio-Rad, Hercules, CA). RT-PCR analysis was carried out with primers designed for human CMO1: 50 -CACAATGGAAAGCAATACCGATATG-30 and 50 -GCAGCTTTTGGGGATCAGTA-30 . Primers for GAPDH (internal standard) were 50 -TG ATGACATCAAGAAGGTGGTGAAG-30 and 50 -TCCTTGGAGGCCATGTA GGCCAT-30 . Amplifications were conducted for 30 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, and extension at 72 °C for 2 min. The PCR products were fractionated on 1.2% agarose gels and photographed using an Alpha-Imager 2000 documentation and analysis system. Real time quantitative RT-PCR Quantitative RT-PCR was carried out in triplicate using a Model 7500 fast real time PCR system and the TaqMan method (Applied BioSystems, Foster City, CA). Primers and probes for human CMO1 (Hs00363176_ml) and 18S rRNA (Hs99999901_sl) were obtained from Applied BioSystems. Relative gene expression was calculated by the DDCt method. Briefly, the resultant mRNA was normalized to a calibrator; in each case, the calibrator chosen was the basal sample. Final results were expressed as n-fold difference in gene expression relative to 18S rRNA and calibrator as follows: n-fold = 2(DCt sampleDCt calibrator), where DCt values of the sample and calibrator were determined by subtracting the average Ct value of the transcript under investigation from the average Ct value of the 18 S rRNA gene for each sample (Applied BioSystems Technical Bulletin No.2). Transient transfections and luciferase assays A549 cells were plated in six-well tissue culture plates at a density of 5 105 cells per well and grown for 24 h, then transfected using Lipofectamin 2000 reagent (Invitrogen) as described previously [24]. Each transfection was performed using 1.0 lg of a luciferase reporter gene plasmid that contained wild-type or sitedirected mutants of the CMO1 basal promoter plus 0.2 lg of an internal control b-galactosidase expression plasmid, pCMV-b-Gal. For co-transfection assays, the total amount of DNA was kept constant using a control vector (pGL3-basic or pcDNA3). One day after transfection, cells were treated for various time points and cell lysates were analyzed for luciferase and b-galactosidase activities. Luciferase activity relative to the pGL3-basic control vector was determined after adjustment for b-galactosidase level. All
transfections were performed in triplicate each in at least three independent experiments.
Electrophoretic mobility shift assays (EMSA) Nuclear extracts were prepared with NE nuclear and cytoplasmic extraction reagent (Sigma) as described previously [24]. Double-stranded oligonucleotides containing sequences from the human CMO1 promoter were synthesized (Operon Biotechnologies, Inc., Huntsville, Ala.). The oligonucleotide sense sequences tested were as follows: wild-type PPAR site, 50 -GCTTGGAAATTAA CCTTTAACCAAACAT-30 and mutant PPAR site, 50 -GCTTGGAAAT TAtgCTTTAtgCAAACAT-30 . (Lower case indicates mutated nucleotides.) EMSA reaction mixtures (20 ll final volumes) included 5–15 lg of nuclear extracts, 25 mM HEPES, 100 mM KCl, 0.1% Nonidet P-40 (v/v), 1 mM dithiothreitol, 5% glycerol and 1 lg of poly (dI-dC) (Sigma) as a nonspecific competitor. After incubation for 20 min on ice, 20 fmol of [c-32P]-ATP end-labeled probe was added and the reaction was incubated for 30 min at room temperature. Protein-DNA complexes were separated on a 5% nondenaturing polyacrylamide gel in 0.5 TBE (Tris–borate-EDTA buffer) at 25 °C. For competition assays, 100-fold molar excess of unlabeled double-stranded oligonucleotide competitor was incubated with the nuclear extract prior to adding the 32P-labeled probe.
Western blot analysis Total cellular protein was isolated at indicated time points using ice-cold M-PER mammalian protein extraction reagent (Pierce, Rockford, IL) containing 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Roche Diagnostics GmbH). Protein concentrations were determined by BCA protein assay (Pierce). Proteins (30–50 lg) were separated by 10% SDS–PAGE and transferred to Immun-blot™ PVDF membranes (Bio-Rad) by semi-dry blotting. Membranes were blocked with 5% non-fat milk dissolved in Tris buffered saline-Tween and probed with the appropriate antibody, followed by incubation with an anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase. Immunoblots were developed using the ECL™ western blotting detection reagents (Amersham Biosciences) and then scanned. Quantification of bands was performed using NIH Image J software.
Indirect immunofluorescence Cells grown on coverslips were fixed in fresh 3.7% paraformaldehyde in PBS for 20 min at room temperature and blocked with 3% BSA in PBS. Specimens were incubated with CMO1 primary antibody in PBS containing 3% BSA, followed by incubation with goat anti-rabbit IgG conjugated with Alexa Fluor 488 (Invitrogen) in PBS with 3% BSA. Cells were examined by fluorescence microscopy (Olympus BX51, Tokyo, Japan).
Statistics Experiments were conducted either in duplication or in triplicate and all experiments were repeated at least three times. Statistical analyses were performed with Student’s t tests for differences between two groups. Data are expressed as mean ± S.D., and p values smaller than 0.05 were considered to be statistically significant.
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Results CMO1 expression in human alveolar epithelial cells Early studies showed that CMO1 mRNA was prominently expressed in the intestine, liver, and kidney as well as lung [14]. A principal site of vitamin A action in the lung is the terminal air sacs [32]. Consequently, we examined CMO1 expression of endogenous CMO1 in a human pulmonary alveolar epithelial cell line (A549) widely utilized to investigate air sac biochemistry, molecular biology and physiology [33,34]. As shown in Fig. 1A, CMO1 mRNA is detected in A549 alveolar epithelial cells. CMO1 protein also is accumulated, verified by western blot analysis of cell lysates (Fig. 1B) and CMO1 cytoplasmic detection by immunofluorescence (Fig. 1C).
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indicates appearance of a new peak with retention time 2.52 min and kmax of 376 nm. It corresponds to all-trans retinal. A549 cell conversion of b-carotene to retinal was concentration- and timedependent (data not shown). Suppression of CMO1 expression by b-carotene We next determined if b-carotene modulates expression of CMO1, its metabolic enzyme, in pulmonary alveolar epithelial cells. Fig. 3A shows treatment of A549 cells with b-carotene (1 lM) for 24 h resulted in a 45% decrease of CMO1 mRNA accumulation. Parallel protein samples assayed by western blot (Fig. 3B) revealed that CMO1 protein levels were decreased by b-carotene to a remarkably similar extent, approximately 50% of untreated cells. In contrast, ATRA exposure for 24 h yielded modest, non-significant increases in CMO1 mRNA and protein.
Metabolism of b-carotene
Reduction of CMO1 promoter activity by b-carotene
We next examined whether A549 cells can metabolize b-carotene to biologically active retinoic acid (RA) isomers. RA production was determined by transfecting cells with a RAR-driven luciferase reporter gene construct (pRARE-200Luc) for 24 h followed by treatment with b-carotene or ATRA (as a positive control) for 12 h. As shown in Fig. 2A, b-carotene (1 lM) treatment resulted in a 2.6-fold increase in RA-dependent transcriptional activity compared with untreated cells. An approximately 4.8-fold luciferase induction was observed in cells treated with an equimolar concentration of ATRA, suggesting relatively efficient carotene-to-RA metabolism. To further confirm cellular b-carotene metabolism to retinal, HPLC retinoid analysis was performed. In these experiments, cells were incubated with b-carotene (3 lM) for 12 h. Cell pellets and medium were collected, lipids extracted and b-carotene, carotenoid metabolites and retinoids were analyzed by HPLC. Fig. 2B (upper panel) contains a major peak at a retention time of 11.83 min, corresponding to authentic b-carotene. The lower panel
As a next step, we determined if b-carotene-mediated reduction of CMO1 expression is transcriptionally controlled. We first examined the effects of b-carotene and ATRA on CMO1 promoter activity. A549 cells were transfected with the basal CMO1 promoter linked to a luciferase reporter gene (pGL3-BCO208-Luc) described previously [24]. Cells were transfected for 24 h and then treated with b-carotene or ATRA for an additional 24 h. As shown in Fig. 4A, b-carotene significantly reduced CMO1 promoter activity compared with vehicle control. In contrast, ATRA only modestly increased CMO1 promoter activity. This observation is concordant with the effects of b-carotene on CMO1 mRNA and protein levels. To establish the time courses of b-carotene and ATRA action, we also transfected the cells with the reporter gene and treated with b-carotene or ATRA for 2, 4, 6, 16 and 24 h. b-Carotene slightly increased CMO1 promoter transactivation at 4–6 h followed by significant inhibition at longer incubation times. This result is consistent with the observation described above that A549 cell conversion of b-carotene to retinal (retinaldehyde) was concentration- and time-dependent. In contrast, ATRA treatment significantly decreased CMO1 transactivation at 6 h, and then modestly increased transactivation by 24 h (Fig. 4B). Suppression of PPARc/RXRa binding to the CMO1 promoter by bcarotene: cis- and trans-acting effects
Fig. 1. Expression of CMO1 in human pulmonary alveolar epithelial cells. (A) Expression of CMO1 mRNA in pulmonary alveolar epithelial A549 cells. Total RNA was isolated from A549 cells and RT-PCR was performed as described in Materials and methods. GAPDH was used as loading control and the three lanes represent triplicate PCR reactions. (B) Expression of CMO1 protein in A549 cells. Lysates from A549 cells (middle, right lanes) and HEK293 cells over-expressing BCO1 as positive control (left lane) were subjected to SDS–PAGE. Western blot analysis was performed using anti-rabbit antibody against CMO1 protein C-terminal peptide. Equal loading was confirmed by stripping the blot and re-probing it with b-actin antibody. (C) Immunostaining of CMO1 in the A549 cells. A549 cells on coverslips were fixed with 3.7% paraformaldehyde in PBS and incubated with CMO1 primary antibody, followed by incubation with goat anti-rabbit IgG conjugated with Alexa Fluor 488. The image (right panel) showed cytoplasmic localization of CMO1, the nuclei (blue) were stained with DAPI. Scale bar = 10 lm.
Gong et al. previously showed the MEF2 and PPAR transcription factors are essential for basal CMO1 expression in humans [24]. To determine if b-carotene inhibits CMO1 gene expression via MEF2 and/or PPAR DNA binding, we mutated these sites in the CMO1 basal promoter. The wild type basal promoter (pGL3-BCO218) construct and two site-specific mutant constructs were transiently transfected and cells were treated with b-carotene or ATRA (Fig. 5A, left panel). As shown in Fig. 5A (right panel), inactivating the MEF2 binding site slightly decreased reporter activity compared with wild-type promoter controls. Addition of b-carotene further inhibited reporter activity but not sufficiently to account for the bulk of the inhibition caused by b-carotene. In contrast, disruption of the PPAR site abolished more than 3=4 ’s of promoter activity independent of the presence of b-carotene or ATRA. These results indicate that the PPRE is the principal locus for b-carotenemediated transcriptional inhibition of CMO1 expression. The above findings prompted a closer examination of b-carotene effects on PPARc/RXRa heterodimer transactivation. In these experiments, we transiently transfected A549 cells with PPARc and RXRa expression vectors as well as the CMO1 reporter plasmid, individually or in combinations. As shown in Fig. 5B, coexpression of PPARc and RXRa in the presence of PPAR agonist
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Fig. 2. All-trans b-carotene metabolism in A549 cells. (A) b-Carotene activates RAR-dependent transcription. A549 cells were transiently transfected with 1 lg of pRARE-200Luc plus 0.2 lg of pCMV-b-Gal vector as a control for transfection efficiency. After 24 h transfection, cells were treated with DMSO (Control), b-carotene (1 lM) and ATRA (1 lM) for 12 h. Luciferase and b-Gal activities were measured and relative fold of luciferase activity was determined after adjustment for b-Gal activity. Results are presented as means ± S.D. of three independent experiments each performed in triplicate, ⁄p < 0.05. (B) (top panel) HPLC chromatogram of b-carotene from 12 h incubation of A549 cells with 3 lM b-carotene as described (with mobile phase A) under ‘‘Methods’’. A peak corresponding to authentic b-carotene was detected with kmax at 450 nm. (Lower panel) HPLC chromatogram of b-carotene cleavage products from 12 h incubation of A549 cells with 3 lM b-carotene (with mobile phase B). Inset: absorption spectra of all-trans bcarotene, b-carotene cleavage product with kmax at 376 nm corresponding to all-trans-retinal with kmax at 380 nm.
was required to induce maximal assay activity. PPARc/RXRa heterodimerization is essential for stimulated CMO1 transcription. To corroborate these results, we investigated the effects of bcarotene and ATRA on PPARc/RXRa heterodimer binding to the basal CMO1 promoter PPRE. A549 cells were treated with b-carotene and ATRA for 24 h and nuclear extracts were isolated for electrophoretic mobility shift assay (EMSA). As shown in Fig. 5C (left panel), the wild type double-stranded probe, when incubated with nuclear extract from A549 cells, forms a single DNA–protein complex. The PPARc/RXRa complex was competed away by a 100-fold excess unlabeled wild-type DNA sequence. In addition, incubating cell nuclear extract with a double-stranded DNA probe containing a mutated PPRE prevented formation of the PPARc/RXRa-DNA complex. Fig. 5C (right panel) shows that pre-treating cells with b-carotene strongly attenuated binding of the PPARc/RXRa complex to PPRE oligonucleotide. ATRA pre-treatment of cells had no
effect on formation of this transcriptionally active protein-DNA complex. CMO1-dependent inhibition of PPARc activity by b-carotene: the effect of retinaldehyde To further verify the effects of b-carotene on PPARc/RXRa complex, we determined the activation of PPRE-tk-luc reporter by cotransfection with constructs for PPARc and RXRa into CMO1 stably expressed HEK293 cells, followed by PPARc agonist (GW1929, 5 lM) stimulation in the presence of b-carotene or retinal (retinaldehyde). As shown in Fig. 6A, both retinal and b-carotene significantly inhibited PPRE activation by GW1929 in the CMO1 wild-type stably expressed HEK293 cells. Although retinal alone had similar inhibitory effects on PPRE activation by the PPARc agonist in both pcDNA3.1 and CMO1-mutant stably expressed
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Fig. 3. Regulation of CMO1 gene expression by b-carotene and ATRA. (A) bCarotene decreases the levels of CMO1 mRNA. A549 cells were treated with vehicle (DMSO), b-carotene (1 lM) and ATRA (1 lM) respectively for 24 h. Quantification of CMO1 transcripts were examined by qRT-PCR. Relative fold accumulation uses 2DDCt. Results are presented as means ± S.D. of three independent experiments, each performed in triplicate. ⁄p < 0.05. (B) CMO1 protein from each treatment was analyzed by Western blot using rabbit anti-CMO1 antibody. b-actin was used as loading control (top panel). The bottom panel indicated average of adjusted CMO1 protein levels (mean ± S.D., n = 3), ⁄p < 0.05.
HEK293 cells, the inhibitory effects of b-carotene on PPRE activation was diminished (Fig. 6B). These experiments indicate that conversion of b-carotene to retinal contributes to the inhibition of PPARc activity. Discussion In humans, provitamin A carotenoids are the major dietary precursors for vitamin A. Human lung physiology is retinoid-dependent, especially for regulation of development, pulmonary alveolar formation, and repair [32,35]. In the present study, we investigated the effects of the principal pro-vitamin A carotenoid, b-carotene, on human alveolar epithelial cell regulation of the bcarotene-to-retinal enzyme, CMO1. The results reveal human lung tissue and alveolar epithelial cells express CMO1 (Fig. 1).
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Fig. 4. Inhibition of CMO1 promoter activity by b-carotene. (A) b-Carotene decreases CMO1 promoter activity. A549 cells were transiently transfected with 1 lg of pGL3-BCO218-Luc plus 0.2 lg of pCMV-b-Gal vector as a control for transfection efficiency. After 16 h transfection, cells were treated with DMSO (Control), b-carotene (1 lM) and ATRA (1 lM) for 24 h. Luciferase and b-Gal activities were measured and relative fold of luciferase activity (compared to pGL3Basic) was determined after adjustment for b-Gal activity. Results are presented as means ± S.D. of three independent experiments each performed in triplicate, ⁄ p < 0.05. (B) Time-dependent regulation of CMO1 promoter activity by b-carotene and ATRA. A549 cells were transfected with pGL3-BCO218-Luc and pCMV-b-Gal and treated with DMSO (Control), b-carotene (1 lM) and ATRA (1 lM) for 2, 4, 6, 16 and 20 h. Luciferase and b-Gal activities were measured and relative fold of luciferase activity (compared to pGL3-Basic) was determined after adjustment for b-Gal activity. Results are presented as means ± S.D. of three independent experiments each performed in triplicate, ⁄p < 0.05.
b-carotene treatment of A549 alveolar epithelial cells results in RA production, read out as a functional assay of RAR activity and as detection of retinal and other retinoid metabolites (Fig. 2). CMO1 expression [15] and CMO1 metabolic activity [36] are present to varying extents in several tissues including the lung in several species examined [19–21] [Gong, Exp Lung Res, in press]. b-Carotene accumulates in several tissues including lung in CMO1 null mice [37]. The present study provides functional evidence that b-carotene can be metabolized to retinoids in human pulmonary alveolar epithelial cells and may contribute to vitamin A-dependent processes in lung development or regeneration. CMO1 expression and activity in gastrointestinal tissues are known to be subject to metabolic regulation. Administration of b-carotene or RA decreases intestinal CMO1 expression and activity [22,25,26]. Recently, a gut-specific homeodomain transcription factor, ISX, was identified as a repressor of intestinal SR-BI and CMO1 expression [25] by which RA and its receptors control ISX expression upstream of CMO gene transcription [26]. Our study indicates b-carotene, but not ATRA, suppresses CMO1 expression in human alveolar epithelial cells at both mRNA and protein levels
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Fig. 5. Reduction of the PPARc/RXRa heterodimer binding to CMO1 promoter by bcarotene. (A) A549 cells were transfected with 1.0 lg/well of pGL3-BCO218-Luc or with the corresponding mutated sequences as indicated. Cells were treated with DMSO (Control), b-carotene (1 lM) and ATRA (1 lM) for 24 h after 16 h of transfection. Luciferase and b-Gal activities were measured and relative fold of luciferase activity (compared to pGL3-basic) was determined after adjustment for b-Gal activity. Results shown are means ± S.D. of three independent experiments each performed in triplicate. ⁄p < 0.05, ⁄⁄p < 0.01. (B) A549 cells were transfected with 0.5 lg/well of pGL3-BCO218-Luc reporter gene and expression vectors for PPARc and RXRa alone or in combination. The total amount of DNA for each transfection was kept constant by using a control vector (pGL3-basic). After 16 h transfection, cells were treated with a PPARc agonist (GW1929) for 6 h. Luciferase and b-Gal activity were measured and relative fold of luciferase activity was determined after adjusting for b-Gal activity. Results are presented as means ± S.D. of three independent experiments each performed in triplicate. ⁄p < 0.05, ⁄⁄p < 0.01. (C) EMSA showing nuclear protein-DNA binding to the PPRE site of the CMO1 promoter. Oligomers were end-labeled with [c-32P]-ATP and incubated with nuclear extracts from A549 cells (see ‘‘Methods’’). Addition of 100-fold molar excess of unlabeled competitor oligomers or mutated probes is indicated above each lane (left panel). [c-32P]-ATP labeled probe was incubated with nuclear extracts from A549 cells treated for 20 h with DMSO, b-carotene (1 lM) and alltrans RA (1 lM) as indicated above each lane (right panel).
Fig. 6. Suppression of the PPARc/RXRa activity by b-carotene in HEK293 cells stably expressing CMO1. (A) CMO1 wild-type stably expressed in HEK293 cells (HEK293-CMO1-wt) were co-transfected with 0.5 lg/well of PPRE-tk-Luc reporter gene and 1.5 lg/well of expression constructs for both PPARc and RXRa. Total amount of DNA for each transfection was kept constant using control vector DNA (pGL-3 basic). After transfection (16 h), cells were treated with DMSO (Control), the PPARc agonist GW1929 (5 lM), b-carotene (1 lM) or retinal (1 lM) for 12 h. Luciferase and b-Gal activities were measured and relative fold of luciferase activity (compared to vehicle control) was determined after adjustment for b-Gal activity. Results shown are means ± S.D. of three independent experiments each performed in triplicate. ⁄p < 0.05, ⁄⁄p < 0.01. (B) CMO1 mutant (HEK293-CMO1-mt) and pcDNA3.1 (HEK293-pcDNA3.1) stably expressed HEK293 cells were co-transfected with 0.5 lg/well of PPRE-tk-Luc reporter gene and 1.5 lg/well of the expression vectors for PPARc and RXRa. The total amount of DNA for each transfection was kept constant by using a control vector (pGL3-basic). After 16 h transfection, cells were treated with DMSO (control), PPARc agonist (GW1929), b-carotene and retinal as indicated for 12 h. Luciferase and b-Gal activity were measured and relative fold of luciferase activity was determined after adjusting for b-Gal activity. Results are presented as means ± S.D. of three independent experiments each performed in triplicate. ⁄p < 0.05, ⁄⁄p < 0.01.
(Fig. 3). This discrepancy might be due to tissue-specific regulation. In peripheral non-gut tissues such as liver [25], over-expression of ISX does not alter CMO1 transcription, suggesting that ISX regulation of CMO1 differs among cell types. Interestingly, we found that cell treatments with b-carotene or ATRA show reciprocal, time-dependent effects on CMO1 promoter activity (Fig. 4B). Equimolar exposure to ATRA initially (at 6 h) inhibited and later (at 24 h) slightly induced CMO1 promoter activity whereas, in contrast, b-carotene at 6 h modestly increased and at 24 h significantly decreased CMO1 promoter activity. These findings could indicate analyte differences with time due
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to increasing b-carotene metabolism, or suggest RA might bi-directionally regulate CMO1 expression, inducing expression at lower concentrations and inhibiting at higher concentrations. The molecular mechanisms warrant further investigation. In a previous study, we demonstrated that human CMO1 is a PPAR target gene that contains a PPAR responsive element that binds PPARc/RXRa heterodimers [24]. In the present study, we showed that b-carotene, but not ATRA, can inhibit CMO1 promoter activity through suppression of PPARc/RXRa heterodimeric binding to the promoter (Fig. 5). Consistent with a b-caroteneinduced reduction of PPARc/RXRa heterodimer formation are observations that b-carotene, but not retinol or RA, represses PPARc expression in adipocytes and inguinal white adipose tissue [37,38]. Moreover, recent studies indicate that retinaldehyde or ATRA can contribute to reduced DNA binding of PPARc/RXRa heterodimers. These effects involve production of retinaldehyde [39] and ATRA from b-carotene, and are blocked by a CMO1 chemical inhibitor [37]. ATRA is a ligand for RARs and PPARb [40,41]. In summary, this study elucidates potential roles for CMO1 expression in alveolar epithelium as a mechanism for local conversion of b-carotene to vitamin A. b-Carotene inhibits CMO1 expression in alveolar epithelial cells through suppression of the binding of the PPARc/RXRa transcriptional complex to the CMO1 promoter and CMO1-dependent inhibition of PPARc activity. CMO1 is a PPARc target gene [24]. Our data indicate that CMO1-dependent effects of b-carotene is mediated by a reduction in binding of PPARc/RXRa complex to the CMO1 promoter and an inhibition of PPARc activity in alveolar epithelial cells. These findings indicate that regulation of CMO1 in alveolar epithelial cells is an important determinant in the crosstalk between the RAR- and PPARc-signaling pathways. Our results may also help to explain, in part, one mechanism that may underlie the increased lung cancer incidence after b-carotene supplementation in smokers during clinical trials (e.g., ATBC [42] and CARET [43]). High doses of b-carotene can inhibit CMO1 expression in alveolar epithelial cells, resulting in b-carotene accumulation and, potentially, pro-oxidant b-carotene metabolites exacerbating oxidant stress in smokers. It is of interest that serum concentrations of b-carotene in these higher risk populations for lung cancer were markedly higher than in the Physician’s Health Study populations [44]. Additionally, studies in the ferret model with smoke exposure have shown that lung tissue concentrations of b-carotene are markedly increased in animals supplemented with b-carotene [45]. These findings suggest that b-carotene metabolism to Vitamin A (ATRA) is impaired in individuals whose carcinogenic exposures put them at higher risk for lung cancer. On the other hand, CMO1 initially cleaves b-carotene to form retinaldehyde which, in turn, suppresses PPARc activity. The PPARc pathway is important in suppression of lung cancer progression. Pharmacological activators of PPARc inhibit growth of non-small cell lung carcinoma (NSCLC) cells and induce apoptosis [46,47]. In human lung tumors, decreased expression of PPARc is correlated with poor prognosis [48] and PPARc overexpression in NSCLC inhibits transformed growth and metastasis and promotes epithelial differentiation [49,50]. Thus, b-carotene regulation of pulmonary CMO1 might be one mechanism by which high dose b-carotene was associated in large clinical trials with increased lung cancer risk in smokers.
work was supported in part by grants from National Institutes of Health (R01-HD42174) and RR18722 to L.P.R. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
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Acknowledgments We thank the late Dr. Stefan Andersson (University of Houston, Houston, Texas) for providing pCMV-BCO1 expression vector. This
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b-Carotene regulates expression of b-carotene 15,150 -monoxygenase in human alveolar epithelial cells Xiaoming Gong a, Raju Marisiddaiah b, Lewis P. Rubin a,⇑ a b
Department of Pediatrics, Texas Tech University Health Science Center, Paul L. Foster School of Medicine, El Paso, TX 79905, USA Children’s Research Institute, St. Petersburg, FL 33701, USA
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Article history: Available online 23 September 2013 Keywords: b-Carotene 15,150 -monooxygenase Retinaldehyde Retinoic acid PPARc RXRa Transcriptional regulation Alveolar epithelium
a b s t r a c t b-Carotene 15,150 -monooxygenase (CMO1, BCMO1) converts b-carotene to retinaldehyde (retinal) and is a key enzyme in vitamin A metabolism. CMO1 activity is robust in the intestine and liver, where cmo1 gene transcription may be subject to negative feedback by accumulation of its metabolic products. Evidence from CMO1 null animals also indicates that non-gastrointestinal CMO1 may be required for tissue-specific conversion of b-carotene into vitamin A. The aim of this study was to investigate the effects of the enzymatic substrate, b-carotene, on regulation of CMO1 in a cell model of human alveolar pneumocytes. We demonstrate that CMO1 is expressed in human alveolar epithelial (A549) cells and converts b-carotene into retinal and biologically active retinoic acids (RA). Exposure to b-carotene suppresses CMO1 expression at both mRNA and protein levels. b-Carotene, but not all-trans RA, decreases CMO1 promoter activity in a time- and dosage-dependent manner. This b-carotene-mediated inhibition of CMO1 expression results from decreased binding of peroxisome proliferator-activated receptor c (PPARc) and retinoid X receptor a (RXRa) in the CMO1 promoter. b-Carotene treatment also antagonizes PPARc activity in HEK293 cells that stably express CMO1 wild-type, but not in cells that express the CMO1 mutant or vector alone. These findings have implications for local vitamin A synthesis in the lung, especially during systemic vitamin A insufficiency and may also help to explain, in part, the mechanism underlying the increased lung cancer risk upon b-carotene supplementation in smokers. Ó 2013 Elsevier Inc. All rights reserved.
Introduction Vitamin A (retinol) is a fat-soluble micronutrient essential for normal morphogenesis and epithelial differentiation and maintenance [1,2]. The importance of vitamin A throughout the life cycle is well established [3] and begins in embryogenesis [3,4]. Vitamin A exerts its biological actions largely through its principal active metabolites, specific retinoic acid (RA) isomers. RA activity, in turn, is mediated by binding to RA receptors (RARa, RARb, RARc) and retinoid X receptors (RXRa, RXRb, RXRc), ligand-inducible transcription factors which are members of the superfamily of nuclear hormone receptors [5]. RARs and RXRs form ligand-dependent heterodimers that bind to regulatory regions in specific target genes and modulate gene transcription [6]. Circulating vitamin A concentrations must be maintained in narrow range in order to avoid either deficiency or toxicity. Consequently, vitamin A production is tightly controlled for normal physiological processes.
⇑ Corresponding author. Address: Department of Pediatrics, Texas Tech University Health Science Center, Paul L. Foster School of Medicine, 4800 Alberta Avenue, El Paso, TX 79905, USA. Fax: +1 915 545 0918. E-mail address: [email protected] (L.P. Rubin). 0003-9861/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2013.09.013
A major dietary source of vitamin A is the pro-vitamin A carotenoids, particularly b-carotene, which is ingested from many vegetables and fruits. In humans, increased b-carotene intake leads to increased plasma concentrations of b-carotene and its metabolites. b,b-carotene 15,150 -monooxygenase (1CMO1, BCMO1) symmetrically cleaves b-carotene to yield two molecules of all-trans retinal (retinaldehyde) [7–10]. Studies undertaken in CMO1 null mice and in human subjects further establish the fundamental role of this enzyme in producing vitamin A aldehyde (retinaldehyde, or retinal) from dietary b-carotene [11,12]. Retinal is converted to retinol, the transport and storage form of vitamin A, and is further oxidized to RA. CMO1 expression and enzyme activity are robust in digestive sites in intestinal mucosa and liver [13] but the enzyme also is expressed in peripheral tissues [14,15]. In humans, unlike rodents, substantial amounts of absorbed b-carotene are not enzymatically cleaved in the intestine [16] and up to 15–30% of absorbed b-carotene remains intact and is delivered to peripheral tissues [17,18].
1 Abbreviations used: CMO1, b-carotene 15,150 -monooxygenase; ATRA, all-trans retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR-response element; EMSA, electrophoretic mobility shift assay.
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Local tissue-specific b-carotene to retinal metabolism may contribute to vitamin A production in extra-intestinal tissues. b-Carotene metabolism and CMO1 activity are present in human and rodent lung tissue [19–21]. However, the molecular details of how b-carotene metabolism in the lung is regulated are largely unknown. Although CMO1 enzymatic biochemistry has been studied for several decades, regulation of CMO1 gene expression and enzyme activity has largely focused on the intestinal tract, where CMO1 is subject to transcriptional feedback inhibition by RA [22]. Intestinal cell CMO1 gene expression in rodents also is stimulated by PPARc agonist and PPARc/RXRa heterodimer binding to a PPAR regulatory element (PPRE) [23]. In human intestinal cells, PPARc is essential but not sufficient to activate human CMO1 gene expression which is instead dependent on cooperation between PPARc and MEF2 isoforms [24]. More recently, an intestine specific homeodomain transcription factor, ISX, has been found to regulate CMO1 and scavenger receptor class B type 1 (SR-BI) expression, also via metabolic feedback [25,26]. Mechanisms of CMO1 regulation in extra-intestinal sites remains less studied. In the present report, we examined the effects of b-carotene on the expression of CMO1 in pulmonary alveolar epithelial cells. Lung terminal air sacs, or alveoli, are an important target for retinoid action, especially in development and during repair. We present evidence that b-carotene, but not ATRA, decreases CMO1 expression and inhibits CMO1 promoter activity through suppression of PPARc/RXRa binding to the CMO1 promoter. Moreover, a CMO1-dependent decrease in PPARc activity by b-carotene appears to be a key determinant in regulation of CMO1 expression. Materials and methods Plasmids and chemicals The plasmid pGL3-basic (Promega, Madison WI) and constructs for wild type, truncated and mutated pGL3-BCO1-Luciferase (pGL3-BCO1-Luc) reporter genes, pPPRE-tk-Luc reporter gene, pRARE-200Luc reporter gene, the expression vectors for PPARa, b, and c, RARb, RXRa, pCMV-b-Gal and pcDNA3.1 were described previously [24]. The pCMV-BCO1 expression vector was the gift of Dr. Stefan Andersson (Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, Texas). Dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), b-carotene, retinaldehyde and all-trans retinoic acid (ATRA) were from Sigma–Aldrich (St. Louis, MO). The PPARc agonist, GW1929, was obtained from Alexis Biochemicals (San Diego, CA). Antibodies The synthetic peptide [Ac-C]TKKQAASEEQRDRASD-Amide, corresponding to the C-terminal 15 amino acid residues 525–540 in human CMO1, was coupled to keyhole limpet hemocyanin and used for immunization of rabbits as described previously [27]. Polyclonal antibodies to CMO1 were characterized by an enzyme-linked immunosorbent assay using the synthetic peptide as antigen and by Western blot analysis.
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fetal bovine serum, were purchased from Invitrogen (Carlsbad, CA). Equal numbers of A549 cells (1 106/well) were plated into six-well cell culture plates for 24 h before addition of various treatments. b-carotene (stock solution, 1 mM) was dissolved in DMSO:THF (4:1). ATRA and retinaldehyde (in DMSO) at a concentration of 1 mM were prepared freshly before each experiment. Control cells were incubated with vehicle alone. Site-directed mutagenesis A panel of mutant CMO1 was made using a pcDNA3-CMO1 construct as template. The 5 residues of human CMO1 under study (His172, His237, His308, Glu450, His514) as described previously [28] were mutated to alanine using QuikChange™ multiple site-directed mutagenesis kit (Stratagene). The validity of all point mutations and integrity of the open reading frame were verified by DNA sequencing. Stable expression of wild-type and mutated CMO1 in HEK293 cells Stable CMO1 wild type (CMO1-wt), mutated (CMO1-mt) and pcDNA3.1 cell lines were established as previously described [29]. Briefly, the pcDNA3.1 vector and pcDNA3.1 plasmids containing the full-length wild type or [H172, H308, E450, H514] CMO1 mutants were transfected into HEK293 cells using Lipofectamin 2000 reagent (Invitrogen) and cells were selected in the presence of geneticin (G418, Gibco-BRL) (400 lg/ml). Isolated clones were selected by limited dilution and then expanded. Stable transfected clones were selected in the presence of G418 (500 lg/ml) and verified with PCR using primers for CMO1 and western blot using CMO1 antibody. Extraction of carotenoids and retinoids b-Carotene and its metabolites and retinoids were extracted as described previously, with minor modifications [30]. After incubation of cells with known amounts of b-carotene for the indicated times, culture plates were placed on ice, medium removed and monolayers washed with 0.5 ml of 10 mM sodium taurocholate in phosphate-buffered saline (PBS) to remove surface-bound carotenoids. After PBS washes 2, cells were harvested by brief trypsinization, cell pellets were homogenized in 0.5 ml ice-cold PBS and transferred it to glass test tubes. An aliquot (0.1 ml from 0.2 mM stock) of butylated hydroxytoluene (BHT) was added to the homogenate and, when required, a known amount of internal standard (echinenone) was added. Homogenates were extracted with 1.5 ml dichloromethane/methanol (1:2, v/v) and hexane. Following centrifugation, the resulting upper hexane-dichloromethane layer was collected. The lower layer was extracted two more times and the hexane-dichloromethane layer was combined with the initial extract. The combined extract was dried, re-dissolved in 0.1 mL dichloromethane/methanol (1:4, v/v) and subjected to HPLC analysis. We also analyzed the concentration of b-carotene in the medium before and after incubations. Sample handling, homogenization and extraction were carried out under cold conditions and dim yellow light to minimize carotenoid isomerization and oxidation.
Cell culture and treatments HPLC analysis of b-carotene and retinoids Human pulmonary alveolar epithelial A549 cells and HEK293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with penicillin (100 units/ ml), streptomycin (100 lg/ml), and 10% fetal bovine serum (HyClone, Logan, UT) at 37 °C in a humidified atmosphere of 95% air/ 5% CO2. All the above-mentioned reagents, with the exception of
b-Carotene and retinoids were analyzed and quantified as described previously, with minor modifications [31]. A Shimadzu HPLC system (Model: UFLC, Kyoto, Japan) equipped with PDA detector, SPD-M20A monitoring from 210 to 670 nm, comprising of a gradient pump system, LC-20AT and a personal computer equipped with LC Solution (Shimadzu) software was used for the
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detection and quantification of b-carotene and retinoids. b-Carotene and carotenoid metabolites were separated on a C30 carotenoid column (5 lm, 4.6 150 mm, YMC; Waters), attached to a guard cartridge (5 lm, 4.0 20 mm, YMC; Waters). Isocratic analysis was performed at a flow rate of 1 ml/min using acetonitrile:methanol:dichloromethane (60:30:10, v/v/v) containing 0.1% ammonium acetate (mobile phase A) using detection at 450 nm for b-carotene and 325, 350 and 376 nm, respectively, for retinol, retinoic acid and retinal. When necessary, the b-carotene cleavage product, retinal, was separated on a C18 column (5 lm, 4.6 150 mm, Biobasic-18; Thermo Scientific) attached to a guard cartridge (5 lm, 4 10 mm, Biobasic-18; Thermo Scientific) using acetonitrile:water (90:10, v/v) containing 0.1% ammonium acetate (mobile phase B) at a flow rate of 1 ml/min isocratically. The individual peak identities were confirmed by their characteristic absorption spectra using a PDA detector and were quantified by individual peak AUC referenced to the respective standard. RNA preparation and semi-quantitative RT-PCR Total RNA was isolated from control and treated A549 cells with TRIzol reagent (Invitrogen) and purified using the RNeasy system (Qiagen). About 1 lg of total RNA was reverse-transcribed using iScript™ cDNA Synthesis (Bio-Rad, Hercules, CA). RT-PCR analysis was carried out with primers designed for human CMO1: 50 -CACAATGGAAAGCAATACCGATATG-30 and 50 -GCAGCTTTTGGGGATCAGTA-30 . Primers for GAPDH (internal standard) were 50 -TG ATGACATCAAGAAGGTGGTGAAG-30 and 50 -TCCTTGGAGGCCATGTA GGCCAT-30 . Amplifications were conducted for 30 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, and extension at 72 °C for 2 min. The PCR products were fractionated on 1.2% agarose gels and photographed using an Alpha-Imager 2000 documentation and analysis system. Real time quantitative RT-PCR Quantitative RT-PCR was carried out in triplicate using a Model 7500 fast real time PCR system and the TaqMan method (Applied BioSystems, Foster City, CA). Primers and probes for human CMO1 (Hs00363176_ml) and 18S rRNA (Hs99999901_sl) were obtained from Applied BioSystems. Relative gene expression was calculated by the DDCt method. Briefly, the resultant mRNA was normalized to a calibrator; in each case, the calibrator chosen was the basal sample. Final results were expressed as n-fold difference in gene expression relative to 18S rRNA and calibrator as follows: n-fold = 2(DCt sampleDCt calibrator), where DCt values of the sample and calibrator were determined by subtracting the average Ct value of the transcript under investigation from the average Ct value of the 18 S rRNA gene for each sample (Applied BioSystems Technical Bulletin No.2). Transient transfections and luciferase assays A549 cells were plated in six-well tissue culture plates at a density of 5 105 cells per well and grown for 24 h, then transfected using Lipofectamin 2000 reagent (Invitrogen) as described previously [24]. Each transfection was performed using 1.0 lg of a luciferase reporter gene plasmid that contained wild-type or sitedirected mutants of the CMO1 basal promoter plus 0.2 lg of an internal control b-galactosidase expression plasmid, pCMV-b-Gal. For co-transfection assays, the total amount of DNA was kept constant using a control vector (pGL3-basic or pcDNA3). One day after transfection, cells were treated for various time points and cell lysates were analyzed for luciferase and b-galactosidase activities. Luciferase activity relative to the pGL3-basic control vector was determined after adjustment for b-galactosidase level. All
transfections were performed in triplicate each in at least three independent experiments.
Electrophoretic mobility shift assays (EMSA) Nuclear extracts were prepared with NE nuclear and cytoplasmic extraction reagent (Sigma) as described previously [24]. Double-stranded oligonucleotides containing sequences from the human CMO1 promoter were synthesized (Operon Biotechnologies, Inc., Huntsville, Ala.). The oligonucleotide sense sequences tested were as follows: wild-type PPAR site, 50 -GCTTGGAAATTAA CCTTTAACCAAACAT-30 and mutant PPAR site, 50 -GCTTGGAAAT TAtgCTTTAtgCAAACAT-30 . (Lower case indicates mutated nucleotides.) EMSA reaction mixtures (20 ll final volumes) included 5–15 lg of nuclear extracts, 25 mM HEPES, 100 mM KCl, 0.1% Nonidet P-40 (v/v), 1 mM dithiothreitol, 5% glycerol and 1 lg of poly (dI-dC) (Sigma) as a nonspecific competitor. After incubation for 20 min on ice, 20 fmol of [c-32P]-ATP end-labeled probe was added and the reaction was incubated for 30 min at room temperature. Protein-DNA complexes were separated on a 5% nondenaturing polyacrylamide gel in 0.5 TBE (Tris–borate-EDTA buffer) at 25 °C. For competition assays, 100-fold molar excess of unlabeled double-stranded oligonucleotide competitor was incubated with the nuclear extract prior to adding the 32P-labeled probe.
Western blot analysis Total cellular protein was isolated at indicated time points using ice-cold M-PER mammalian protein extraction reagent (Pierce, Rockford, IL) containing 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Roche Diagnostics GmbH). Protein concentrations were determined by BCA protein assay (Pierce). Proteins (30–50 lg) were separated by 10% SDS–PAGE and transferred to Immun-blot™ PVDF membranes (Bio-Rad) by semi-dry blotting. Membranes were blocked with 5% non-fat milk dissolved in Tris buffered saline-Tween and probed with the appropriate antibody, followed by incubation with an anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase. Immunoblots were developed using the ECL™ western blotting detection reagents (Amersham Biosciences) and then scanned. Quantification of bands was performed using NIH Image J software.
Indirect immunofluorescence Cells grown on coverslips were fixed in fresh 3.7% paraformaldehyde in PBS for 20 min at room temperature and blocked with 3% BSA in PBS. Specimens were incubated with CMO1 primary antibody in PBS containing 3% BSA, followed by incubation with goat anti-rabbit IgG conjugated with Alexa Fluor 488 (Invitrogen) in PBS with 3% BSA. Cells were examined by fluorescence microscopy (Olympus BX51, Tokyo, Japan).
Statistics Experiments were conducted either in duplication or in triplicate and all experiments were repeated at least three times. Statistical analyses were performed with Student’s t tests for differences between two groups. Data are expressed as mean ± S.D., and p values smaller than 0.05 were considered to be statistically significant.
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Results CMO1 expression in human alveolar epithelial cells Early studies showed that CMO1 mRNA was prominently expressed in the intestine, liver, and kidney as well as lung [14]. A principal site of vitamin A action in the lung is the terminal air sacs [32]. Consequently, we examined CMO1 expression of endogenous CMO1 in a human pulmonary alveolar epithelial cell line (A549) widely utilized to investigate air sac biochemistry, molecular biology and physiology [33,34]. As shown in Fig. 1A, CMO1 mRNA is detected in A549 alveolar epithelial cells. CMO1 protein also is accumulated, verified by western blot analysis of cell lysates (Fig. 1B) and CMO1 cytoplasmic detection by immunofluorescence (Fig. 1C).
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indicates appearance of a new peak with retention time 2.52 min and kmax of 376 nm. It corresponds to all-trans retinal. A549 cell conversion of b-carotene to retinal was concentration- and timedependent (data not shown). Suppression of CMO1 expression by b-carotene We next determined if b-carotene modulates expression of CMO1, its metabolic enzyme, in pulmonary alveolar epithelial cells. Fig. 3A shows treatment of A549 cells with b-carotene (1 lM) for 24 h resulted in a 45% decrease of CMO1 mRNA accumulation. Parallel protein samples assayed by western blot (Fig. 3B) revealed that CMO1 protein levels were decreased by b-carotene to a remarkably similar extent, approximately 50% of untreated cells. In contrast, ATRA exposure for 24 h yielded modest, non-significant increases in CMO1 mRNA and protein.
Metabolism of b-carotene
Reduction of CMO1 promoter activity by b-carotene
We next examined whether A549 cells can metabolize b-carotene to biologically active retinoic acid (RA) isomers. RA production was determined by transfecting cells with a RAR-driven luciferase reporter gene construct (pRARE-200Luc) for 24 h followed by treatment with b-carotene or ATRA (as a positive control) for 12 h. As shown in Fig. 2A, b-carotene (1 lM) treatment resulted in a 2.6-fold increase in RA-dependent transcriptional activity compared with untreated cells. An approximately 4.8-fold luciferase induction was observed in cells treated with an equimolar concentration of ATRA, suggesting relatively efficient carotene-to-RA metabolism. To further confirm cellular b-carotene metabolism to retinal, HPLC retinoid analysis was performed. In these experiments, cells were incubated with b-carotene (3 lM) for 12 h. Cell pellets and medium were collected, lipids extracted and b-carotene, carotenoid metabolites and retinoids were analyzed by HPLC. Fig. 2B (upper panel) contains a major peak at a retention time of 11.83 min, corresponding to authentic b-carotene. The lower panel
As a next step, we determined if b-carotene-mediated reduction of CMO1 expression is transcriptionally controlled. We first examined the effects of b-carotene and ATRA on CMO1 promoter activity. A549 cells were transfected with the basal CMO1 promoter linked to a luciferase reporter gene (pGL3-BCO208-Luc) described previously [24]. Cells were transfected for 24 h and then treated with b-carotene or ATRA for an additional 24 h. As shown in Fig. 4A, b-carotene significantly reduced CMO1 promoter activity compared with vehicle control. In contrast, ATRA only modestly increased CMO1 promoter activity. This observation is concordant with the effects of b-carotene on CMO1 mRNA and protein levels. To establish the time courses of b-carotene and ATRA action, we also transfected the cells with the reporter gene and treated with b-carotene or ATRA for 2, 4, 6, 16 and 24 h. b-Carotene slightly increased CMO1 promoter transactivation at 4–6 h followed by significant inhibition at longer incubation times. This result is consistent with the observation described above that A549 cell conversion of b-carotene to retinal (retinaldehyde) was concentration- and time-dependent. In contrast, ATRA treatment significantly decreased CMO1 transactivation at 6 h, and then modestly increased transactivation by 24 h (Fig. 4B). Suppression of PPARc/RXRa binding to the CMO1 promoter by bcarotene: cis- and trans-acting effects
Fig. 1. Expression of CMO1 in human pulmonary alveolar epithelial cells. (A) Expression of CMO1 mRNA in pulmonary alveolar epithelial A549 cells. Total RNA was isolated from A549 cells and RT-PCR was performed as described in Materials and methods. GAPDH was used as loading control and the three lanes represent triplicate PCR reactions. (B) Expression of CMO1 protein in A549 cells. Lysates from A549 cells (middle, right lanes) and HEK293 cells over-expressing BCO1 as positive control (left lane) were subjected to SDS–PAGE. Western blot analysis was performed using anti-rabbit antibody against CMO1 protein C-terminal peptide. Equal loading was confirmed by stripping the blot and re-probing it with b-actin antibody. (C) Immunostaining of CMO1 in the A549 cells. A549 cells on coverslips were fixed with 3.7% paraformaldehyde in PBS and incubated with CMO1 primary antibody, followed by incubation with goat anti-rabbit IgG conjugated with Alexa Fluor 488. The image (right panel) showed cytoplasmic localization of CMO1, the nuclei (blue) were stained with DAPI. Scale bar = 10 lm.
Gong et al. previously showed the MEF2 and PPAR transcription factors are essential for basal CMO1 expression in humans [24]. To determine if b-carotene inhibits CMO1 gene expression via MEF2 and/or PPAR DNA binding, we mutated these sites in the CMO1 basal promoter. The wild type basal promoter (pGL3-BCO218) construct and two site-specific mutant constructs were transiently transfected and cells were treated with b-carotene or ATRA (Fig. 5A, left panel). As shown in Fig. 5A (right panel), inactivating the MEF2 binding site slightly decreased reporter activity compared with wild-type promoter controls. Addition of b-carotene further inhibited reporter activity but not sufficiently to account for the bulk of the inhibition caused by b-carotene. In contrast, disruption of the PPAR site abolished more than 3=4 ’s of promoter activity independent of the presence of b-carotene or ATRA. These results indicate that the PPRE is the principal locus for b-carotenemediated transcriptional inhibition of CMO1 expression. The above findings prompted a closer examination of b-carotene effects on PPARc/RXRa heterodimer transactivation. In these experiments, we transiently transfected A549 cells with PPARc and RXRa expression vectors as well as the CMO1 reporter plasmid, individually or in combinations. As shown in Fig. 5B, coexpression of PPARc and RXRa in the presence of PPAR agonist
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Fig. 2. All-trans b-carotene metabolism in A549 cells. (A) b-Carotene activates RAR-dependent transcription. A549 cells were transiently transfected with 1 lg of pRARE-200Luc plus 0.2 lg of pCMV-b-Gal vector as a control for transfection efficiency. After 24 h transfection, cells were treated with DMSO (Control), b-carotene (1 lM) and ATRA (1 lM) for 12 h. Luciferase and b-Gal activities were measured and relative fold of luciferase activity was determined after adjustment for b-Gal activity. Results are presented as means ± S.D. of three independent experiments each performed in triplicate, ⁄p < 0.05. (B) (top panel) HPLC chromatogram of b-carotene from 12 h incubation of A549 cells with 3 lM b-carotene as described (with mobile phase A) under ‘‘Methods’’. A peak corresponding to authentic b-carotene was detected with kmax at 450 nm. (Lower panel) HPLC chromatogram of b-carotene cleavage products from 12 h incubation of A549 cells with 3 lM b-carotene (with mobile phase B). Inset: absorption spectra of all-trans bcarotene, b-carotene cleavage product with kmax at 376 nm corresponding to all-trans-retinal with kmax at 380 nm.
was required to induce maximal assay activity. PPARc/RXRa heterodimerization is essential for stimulated CMO1 transcription. To corroborate these results, we investigated the effects of bcarotene and ATRA on PPARc/RXRa heterodimer binding to the basal CMO1 promoter PPRE. A549 cells were treated with b-carotene and ATRA for 24 h and nuclear extracts were isolated for electrophoretic mobility shift assay (EMSA). As shown in Fig. 5C (left panel), the wild type double-stranded probe, when incubated with nuclear extract from A549 cells, forms a single DNA–protein complex. The PPARc/RXRa complex was competed away by a 100-fold excess unlabeled wild-type DNA sequence. In addition, incubating cell nuclear extract with a double-stranded DNA probe containing a mutated PPRE prevented formation of the PPARc/RXRa-DNA complex. Fig. 5C (right panel) shows that pre-treating cells with b-carotene strongly attenuated binding of the PPARc/RXRa complex to PPRE oligonucleotide. ATRA pre-treatment of cells had no
effect on formation of this transcriptionally active protein-DNA complex. CMO1-dependent inhibition of PPARc activity by b-carotene: the effect of retinaldehyde To further verify the effects of b-carotene on PPARc/RXRa complex, we determined the activation of PPRE-tk-luc reporter by cotransfection with constructs for PPARc and RXRa into CMO1 stably expressed HEK293 cells, followed by PPARc agonist (GW1929, 5 lM) stimulation in the presence of b-carotene or retinal (retinaldehyde). As shown in Fig. 6A, both retinal and b-carotene significantly inhibited PPRE activation by GW1929 in the CMO1 wild-type stably expressed HEK293 cells. Although retinal alone had similar inhibitory effects on PPRE activation by the PPARc agonist in both pcDNA3.1 and CMO1-mutant stably expressed
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Fig. 3. Regulation of CMO1 gene expression by b-carotene and ATRA. (A) bCarotene decreases the levels of CMO1 mRNA. A549 cells were treated with vehicle (DMSO), b-carotene (1 lM) and ATRA (1 lM) respectively for 24 h. Quantification of CMO1 transcripts were examined by qRT-PCR. Relative fold accumulation uses 2DDCt. Results are presented as means ± S.D. of three independent experiments, each performed in triplicate. ⁄p < 0.05. (B) CMO1 protein from each treatment was analyzed by Western blot using rabbit anti-CMO1 antibody. b-actin was used as loading control (top panel). The bottom panel indicated average of adjusted CMO1 protein levels (mean ± S.D., n = 3), ⁄p < 0.05.
HEK293 cells, the inhibitory effects of b-carotene on PPRE activation was diminished (Fig. 6B). These experiments indicate that conversion of b-carotene to retinal contributes to the inhibition of PPARc activity. Discussion In humans, provitamin A carotenoids are the major dietary precursors for vitamin A. Human lung physiology is retinoid-dependent, especially for regulation of development, pulmonary alveolar formation, and repair [32,35]. In the present study, we investigated the effects of the principal pro-vitamin A carotenoid, b-carotene, on human alveolar epithelial cell regulation of the bcarotene-to-retinal enzyme, CMO1. The results reveal human lung tissue and alveolar epithelial cells express CMO1 (Fig. 1).
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Fig. 4. Inhibition of CMO1 promoter activity by b-carotene. (A) b-Carotene decreases CMO1 promoter activity. A549 cells were transiently transfected with 1 lg of pGL3-BCO218-Luc plus 0.2 lg of pCMV-b-Gal vector as a control for transfection efficiency. After 16 h transfection, cells were treated with DMSO (Control), b-carotene (1 lM) and ATRA (1 lM) for 24 h. Luciferase and b-Gal activities were measured and relative fold of luciferase activity (compared to pGL3Basic) was determined after adjustment for b-Gal activity. Results are presented as means ± S.D. of three independent experiments each performed in triplicate, ⁄ p < 0.05. (B) Time-dependent regulation of CMO1 promoter activity by b-carotene and ATRA. A549 cells were transfected with pGL3-BCO218-Luc and pCMV-b-Gal and treated with DMSO (Control), b-carotene (1 lM) and ATRA (1 lM) for 2, 4, 6, 16 and 20 h. Luciferase and b-Gal activities were measured and relative fold of luciferase activity (compared to pGL3-Basic) was determined after adjustment for b-Gal activity. Results are presented as means ± S.D. of three independent experiments each performed in triplicate, ⁄p < 0.05.
b-carotene treatment of A549 alveolar epithelial cells results in RA production, read out as a functional assay of RAR activity and as detection of retinal and other retinoid metabolites (Fig. 2). CMO1 expression [15] and CMO1 metabolic activity [36] are present to varying extents in several tissues including the lung in several species examined [19–21] [Gong, Exp Lung Res, in press]. b-Carotene accumulates in several tissues including lung in CMO1 null mice [37]. The present study provides functional evidence that b-carotene can be metabolized to retinoids in human pulmonary alveolar epithelial cells and may contribute to vitamin A-dependent processes in lung development or regeneration. CMO1 expression and activity in gastrointestinal tissues are known to be subject to metabolic regulation. Administration of b-carotene or RA decreases intestinal CMO1 expression and activity [22,25,26]. Recently, a gut-specific homeodomain transcription factor, ISX, was identified as a repressor of intestinal SR-BI and CMO1 expression [25] by which RA and its receptors control ISX expression upstream of CMO gene transcription [26]. Our study indicates b-carotene, but not ATRA, suppresses CMO1 expression in human alveolar epithelial cells at both mRNA and protein levels
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Fig. 5. Reduction of the PPARc/RXRa heterodimer binding to CMO1 promoter by bcarotene. (A) A549 cells were transfected with 1.0 lg/well of pGL3-BCO218-Luc or with the corresponding mutated sequences as indicated. Cells were treated with DMSO (Control), b-carotene (1 lM) and ATRA (1 lM) for 24 h after 16 h of transfection. Luciferase and b-Gal activities were measured and relative fold of luciferase activity (compared to pGL3-basic) was determined after adjustment for b-Gal activity. Results shown are means ± S.D. of three independent experiments each performed in triplicate. ⁄p < 0.05, ⁄⁄p < 0.01. (B) A549 cells were transfected with 0.5 lg/well of pGL3-BCO218-Luc reporter gene and expression vectors for PPARc and RXRa alone or in combination. The total amount of DNA for each transfection was kept constant by using a control vector (pGL3-basic). After 16 h transfection, cells were treated with a PPARc agonist (GW1929) for 6 h. Luciferase and b-Gal activity were measured and relative fold of luciferase activity was determined after adjusting for b-Gal activity. Results are presented as means ± S.D. of three independent experiments each performed in triplicate. ⁄p < 0.05, ⁄⁄p < 0.01. (C) EMSA showing nuclear protein-DNA binding to the PPRE site of the CMO1 promoter. Oligomers were end-labeled with [c-32P]-ATP and incubated with nuclear extracts from A549 cells (see ‘‘Methods’’). Addition of 100-fold molar excess of unlabeled competitor oligomers or mutated probes is indicated above each lane (left panel). [c-32P]-ATP labeled probe was incubated with nuclear extracts from A549 cells treated for 20 h with DMSO, b-carotene (1 lM) and alltrans RA (1 lM) as indicated above each lane (right panel).
Fig. 6. Suppression of the PPARc/RXRa activity by b-carotene in HEK293 cells stably expressing CMO1. (A) CMO1 wild-type stably expressed in HEK293 cells (HEK293-CMO1-wt) were co-transfected with 0.5 lg/well of PPRE-tk-Luc reporter gene and 1.5 lg/well of expression constructs for both PPARc and RXRa. Total amount of DNA for each transfection was kept constant using control vector DNA (pGL-3 basic). After transfection (16 h), cells were treated with DMSO (Control), the PPARc agonist GW1929 (5 lM), b-carotene (1 lM) or retinal (1 lM) for 12 h. Luciferase and b-Gal activities were measured and relative fold of luciferase activity (compared to vehicle control) was determined after adjustment for b-Gal activity. Results shown are means ± S.D. of three independent experiments each performed in triplicate. ⁄p < 0.05, ⁄⁄p < 0.01. (B) CMO1 mutant (HEK293-CMO1-mt) and pcDNA3.1 (HEK293-pcDNA3.1) stably expressed HEK293 cells were co-transfected with 0.5 lg/well of PPRE-tk-Luc reporter gene and 1.5 lg/well of the expression vectors for PPARc and RXRa. The total amount of DNA for each transfection was kept constant by using a control vector (pGL3-basic). After 16 h transfection, cells were treated with DMSO (control), PPARc agonist (GW1929), b-carotene and retinal as indicated for 12 h. Luciferase and b-Gal activity were measured and relative fold of luciferase activity was determined after adjusting for b-Gal activity. Results are presented as means ± S.D. of three independent experiments each performed in triplicate. ⁄p < 0.05, ⁄⁄p < 0.01.
(Fig. 3). This discrepancy might be due to tissue-specific regulation. In peripheral non-gut tissues such as liver [25], over-expression of ISX does not alter CMO1 transcription, suggesting that ISX regulation of CMO1 differs among cell types. Interestingly, we found that cell treatments with b-carotene or ATRA show reciprocal, time-dependent effects on CMO1 promoter activity (Fig. 4B). Equimolar exposure to ATRA initially (at 6 h) inhibited and later (at 24 h) slightly induced CMO1 promoter activity whereas, in contrast, b-carotene at 6 h modestly increased and at 24 h significantly decreased CMO1 promoter activity. These findings could indicate analyte differences with time due
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to increasing b-carotene metabolism, or suggest RA might bi-directionally regulate CMO1 expression, inducing expression at lower concentrations and inhibiting at higher concentrations. The molecular mechanisms warrant further investigation. In a previous study, we demonstrated that human CMO1 is a PPAR target gene that contains a PPAR responsive element that binds PPARc/RXRa heterodimers [24]. In the present study, we showed that b-carotene, but not ATRA, can inhibit CMO1 promoter activity through suppression of PPARc/RXRa heterodimeric binding to the promoter (Fig. 5). Consistent with a b-caroteneinduced reduction of PPARc/RXRa heterodimer formation are observations that b-carotene, but not retinol or RA, represses PPARc expression in adipocytes and inguinal white adipose tissue [37,38]. Moreover, recent studies indicate that retinaldehyde or ATRA can contribute to reduced DNA binding of PPARc/RXRa heterodimers. These effects involve production of retinaldehyde [39] and ATRA from b-carotene, and are blocked by a CMO1 chemical inhibitor [37]. ATRA is a ligand for RARs and PPARb [40,41]. In summary, this study elucidates potential roles for CMO1 expression in alveolar epithelium as a mechanism for local conversion of b-carotene to vitamin A. b-Carotene inhibits CMO1 expression in alveolar epithelial cells through suppression of the binding of the PPARc/RXRa transcriptional complex to the CMO1 promoter and CMO1-dependent inhibition of PPARc activity. CMO1 is a PPARc target gene [24]. Our data indicate that CMO1-dependent effects of b-carotene is mediated by a reduction in binding of PPARc/RXRa complex to the CMO1 promoter and an inhibition of PPARc activity in alveolar epithelial cells. These findings indicate that regulation of CMO1 in alveolar epithelial cells is an important determinant in the crosstalk between the RAR- and PPARc-signaling pathways. Our results may also help to explain, in part, one mechanism that may underlie the increased lung cancer incidence after b-carotene supplementation in smokers during clinical trials (e.g., ATBC [42] and CARET [43]). High doses of b-carotene can inhibit CMO1 expression in alveolar epithelial cells, resulting in b-carotene accumulation and, potentially, pro-oxidant b-carotene metabolites exacerbating oxidant stress in smokers. It is of interest that serum concentrations of b-carotene in these higher risk populations for lung cancer were markedly higher than in the Physician’s Health Study populations [44]. Additionally, studies in the ferret model with smoke exposure have shown that lung tissue concentrations of b-carotene are markedly increased in animals supplemented with b-carotene [45]. These findings suggest that b-carotene metabolism to Vitamin A (ATRA) is impaired in individuals whose carcinogenic exposures put them at higher risk for lung cancer. On the other hand, CMO1 initially cleaves b-carotene to form retinaldehyde which, in turn, suppresses PPARc activity. The PPARc pathway is important in suppression of lung cancer progression. Pharmacological activators of PPARc inhibit growth of non-small cell lung carcinoma (NSCLC) cells and induce apoptosis [46,47]. In human lung tumors, decreased expression of PPARc is correlated with poor prognosis [48] and PPARc overexpression in NSCLC inhibits transformed growth and metastasis and promotes epithelial differentiation [49,50]. Thus, b-carotene regulation of pulmonary CMO1 might be one mechanism by which high dose b-carotene was associated in large clinical trials with increased lung cancer risk in smokers.
work was supported in part by grants from National Institutes of Health (R01-HD42174) and RR18722 to L.P.R. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
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Acknowledgments We thank the late Dr. Stefan Andersson (University of Houston, Houston, Texas) for providing pCMV-BCO1 expression vector. This
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