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Asymmetric Cleavage of ß-Carotene Yields a Transcriptional Repressor of Retinoid X Receptor and Peroxisome Proliferator-Activated Receptor Responses

Cardiovascular Division (O.Z., G.O., G.S., E.L., J.P.), Brigham and Women’s Hospital, Harvard University, Boston, Massachusetts 02115; Merck Research Laboratories (J.P.B.), Rahway, New Jersey; Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University (G.T., N.I.K., G.G.D.), and Department of Biochemistry (N.I.K.), Tufts University School of Medicine, Boston, Massachusetts 02111

Address all correspondence and requests for reprints to: Jorge Plutzky, M.D., Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, NRB 742D, Boston, Massachusetts 02115. E-mail: jplutzky{at}rics.bwh.harvard.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
ß-Carotene and its metabolites exert a broad range of effects, in part by regulating transcriptional responses through specific nuclear receptor activation. Symmetric cleavage of ß-carotene can yield 9-cis retinoic acid (9-cisRA), the natural ligand for the nuclear receptor RXR, the obligate heterodimeric partner for numerous nuclear receptor family members. A significant portion of ß-carotene can also undergo asymmetric cleavage to yield apocarotenals, a series of poorly understood naturally occurring molecules whose biologic role, including their transcriptional effects, remains essentially unknown. We show here that ß-apo-14'-carotenal (apo14), but not other structurally related apocarotenals, represses peroxisome proliferator-activated receptors (PPAR) and RXR activation and biologic responses induced by their respective agonists both in vitro and in vivo. During adipocyte differentiation, apo14 inhibited PPAR target gene expression and adipogenesis, even in the presence of the potent PPAR agonist BRL49653. Apo14 also suppressed known PPAR responses, including target gene expression and its known antiinflammatory effects, but not if PPAR agonist stimulation occurred before apo14 exposure and not in PPAR-deficient cells or mice. Other apocarotenals tested had none of these effects. These data extend current views of ß-carotene metabolism to include specific apocarotenals as possible biologically active mediators and identify apo14 as a possible template for designing PPAR and RXR modulators and better understanding modulation of nuclear receptor activation. These results also suggest a novel model of molecular endocrinology in which metabolism of a parent compound, ß-carotene, may alternatively activate (9-cisRA) or inhibit (apo14) specific nuclear receptor responses.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
ß-CAROTENE IS A natural compound characterized by a broad range of biologic functions that are essential for both plant and animal metabolism (1, 2). These multiple actions of ß-carotene derive at least in part from its unique structure, which consists of eight isoprenoid units with their associated polyunsaturated double bonds (1, 3). These facets underlie ß-carotene’s capacity for isomerization, singlet oxygen quenching, and electron transfer during oxidation/reduction reactions (1, 3). In terms of metabolism, cleavage of ß-carotene yields an array of transcriptionally active compounds that retain these isomerization and oxidation/reduction capabilities (4). Central cleavage of ß-carotene, a major pathway for its metabolism, generates retinal, vitamin A (retinol), and retinoic acid (RA, Fig. 1; and Refs. 5, 6, 7).

View larger version (21K): [in this window] [in a new window]   Fig. 1. Symmetric and Asymmetric Cleavage of ß-Carotene Generates Various Metabolites that Regulate Nuclear Receptor Activity Symmetric cleavage of ß-carotene produces two retinaldehyde molecules, which can activate the RAR receptor. Enzymatic oxidation of retinaldehyde can produce retinoic acid (RA), a known agonist for RAR and RXR, the latter through 9-cis RA isomers. RA activation of PPAR has also been reported (52 ). Enzymatic reduction of retinaldehyde can generate retinol, another reported RAR activator. Asymmetric cleavage of ß-carotene yields apocarotenals, a specific series of molecules thought to be generated by mitochondrial, enzymatic, or auto-oxidative processes (3 20 22 23 24 ). The biologic role of apocarotenals remains poorly understood; modulation of nuclear receptor responses by apocarotenals has not been previously described.

Adipogenesis is a biologic process dependent on the coordinate regulation of multiple steroid hormone nuclear receptors including RXR (12). For example, whereas intact PPAR activation is essential for adipogenesis both in vitro and in vivo (13), RAR activation inhibits adipocyte differentiation and lipid accumulation (14). All stages of adipogenesis require the participation of RXR heterodimers including early (VDR), intermediate (RXR), and late markers [PPAR, liver X receptor (LXR), and RXR] (12), further highlighting the importance of RXR activation. Much of the insight into the effects of various nuclear receptors on adipogenesis has come through studies with rexinoids, synthetic retinoid-like compounds that can be either retinoid receptor agonists or antagonists (15, 16, 17). Consistent with this, synthetic RXR antagonists decrease adipogenesis (18). Both retinoids and rexinoids have also been implicated in regulating other steroid hormone receptors, for example both RA and the synthetic RA analog CS018 have been reported as ligands for PPAR and PPAR, respectively (19).

The effects of these retinoid receptors and their synthetic modulators underscore the importance of natural retinoids and their endogenous formation. One major pathway for the generation of the natural RXR ligand 9-cis RA is through central cleavage of ß-carotene, which initially yields retinaldehyde before subsequent oxidation and isomerization produces 9-cis RA (Fig. 1; and Refs. 6, 7, 20 and 21). ß-Carotene can also undergo asymmetric cleavage, leading to the formation of a class of molecules known as apocarotenals (Fig. 1; and Refs. 22 and 23). Apocarotenals can be generated in vitro and in vivo, mostly under oxidative conditions and involving the action of specific enzymes such as lipoxygenases and dioxygenases (3, 22, 23, 24). Despite intense study of many ß-carotene-derived retinoid metabolites and their role in transcription (25), little is known about apocarotenals, their biologic role, or their effects on gene expression. We hypothesized that apocarotenals might be biologically active mediators of transcription through effects on nuclear receptor responses. We present evidence here that a specific product of asymmetric ß-carotene cleavage, ß-apo-14'-carotenal (apo14), but not other closely related apocarotenals, interacts with RXR, PPAR, and PPAR to oppose known effects of both synthetic and natural ligands to these receptors, including target gene induction in vitro and functional cellular responses in vivo. The effects of apo14 in selectively inhibiting nuclear receptor responses underscores how distinct ß-carotene metabolites can direct divergent biologic responses, identifies a molecular basis for further testing the physical interaction between nuclear receptors and their modulators and provides a novel template for developing synthetic RXR/PPAR modulators.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Apo14 Inhibits Adipogenesis in Vitro
Given the impact of ß-carotene and its metabolites on adipogenesis (26), we tested the effects of apocarotenals on standard 3T3-L1 adipocyte differentiation assays, focusing initially on apocarotenals with the shortest (apo14) and longest (apo8) chain length. Apo14 inhibited adipogenesis as shown by oil red-O staining of 3T3-L1 preadipocytes stimulated with apo14 (1 µM) once during a 7-d differentiation period (1 x 7 d, Fig. 2A); these apo14 effects on adipogenesis were even more pronounced after repeating administration of apo14 three times during the 7-d differentiation experiment (3 x 7 d, Fig. 2A). Moreover, apo14 decreased adipogenesis even in the presence of the synthetic PPAR agonist BRL49653 (0.5 µM), a potent adipogenic stimulant (Fig. 2A, lower panels). In contrast, despite its structural similarity to apo14, apo8 had no effect on adipogenesis even up to a concentration of 10 µM, in either the presence (Fig. 2B) or absence (data not shown) of BRL (10 µM). Apo14 decreased lipid accumulation during 3T3-L1 in a concentration-dependent manner (0.1–10 µM, data not shown).

View larger version (87K): [in this window] [in a new window]   Fig. 2. Apo14, But Not Other Apocarotenals, Inhibits Adipogenesis and PPAR Target Gene Expression A, Apo14 inhibited lipid accumulation during 3T3-L1 preadipocyte differentiation in either the absence (upper panel) or presence (lower panel) of the PPAR agonist BRL49653 (BRL). Briefly, 3T3-L1 preadipocytes were grown until near confluence before adipogenesis was induced using a standard differentiation mixture (differentiated; see Materials and Methods). After initial differentiation (15 h, d 0), apo14 (1 µM) either alone or in the presence of BRL (0.5 µM) was added once to the medium and differentiation continued for 7 d (middle panel, 1 x 7 d) before intracellular lipid accumulation was detected using oil-red-O staining. Apo8 had no effect on adipogenesis when used in parallel similar studies (data not shown). Adipogenesis assays were repeated but after adding apo14 (1 µM) three times during differentiation, specifically every 48 h (d 0, 2, 6; right panel, 3 x 7 d). After 7 d of differentiation, oil-red-O staining was performed. B, 3T3-L1 preadipocyte differentiation in the presence of BRL (10 µM) was repeated as above but in the presence of either apo14 or apo8 (both 10 µM, added once at d 0, 1 x 7 d). As before, apo14 decreased lipid accumulation, whereas apo8 had no effect, as indicated by oil-red-O staining. Veh, Vehicle. Single administration of apo14 (10 µM, d 0) during 3T3-L1 preadipocyte differentiation inhibited aP2 mRNA expression in a concentration-dependent manner in either the absence (left) or presence (right) of BRL (10 µM), as shown by Northern blot analysis. In contrast, under similar conditions, apo8 stimulation had no effect on aP2 expression at either 10 µM (left panel) or 20 µM (right panel). D, Apo14 inhibits expression of adiponectin (ADPN) in a concentration-dependent manner during 3T3-L1 differentiation in the presence of BRL (1 µM) using similar protocols as before (single apo14 administration, d 0), with effects evident at an apo14 concentration of 100 nM.

Apo14 Suppresses RXR, PPAR, and PPAR-Ligand Binding Domain (LBD) Activation
Because some retinoids can activate RAR, a known suppressor of adipogenesis (27), we asked whether RAR activation might account for the decreased adipogenesis seen with apo14. RAR-LBD activation was studied using established human LBD-yeast GAL4 transfection assays in bovine aortic endothelial cells (ECs) as before (28). Apo14, apo8, and ß-carotene only modestly activated RAR, even at concentrations up to 10 µM, with significantly lesser effects than those induced by specific synthetic (TTNPB) and natural (all-trans RA) RAR ligands (white bars, Fig. 3A). In concentration-dependent experiments in either the absence or presence of TTNPB, apo14 again only modestly activated RAR; moreover, in cells pretreated with TTNPB, apo14 modestly inhibited RAR activation (Fig. 3B). Whereas these data indicate that apo14 may be a weak partial RAR agonist, it also suggests that RAR-independent mechanisms may contribute to apo14’s potent repression of adipogenesis. Indeed, concentrations of apo14 that blocked adipogenesis (<1 µM) had no effect on the RAR-LBD or its activation by TTNPB (Supplemental Fig. 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). These data suggest apo14 may inhibit adipogenesis in a manner distinct from other retinoids and their RAR activation.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Apo14, But Not Other Apocarotenals, Selectively Inhibits RAR, RXR, and PPAR Activation by their Respective Agonists A, Apo14 inhibits RAR-LBD activation by either natural or synthetic RAR agonists. Standard LBD-GAL4 assays using human RAR LBD, as well as the other LBDs tested below, were performed in primary bovine ECs (BAECs) as previously described (40 ). Cells were stimulated with ß-carotene (1 µM) or apo14, apo12, apo8 (all at 10 µM, 18 h) or vehicle to test for RAR activation or inhibition, the latter involving preincubation of cells with the same concentration of apocarotenals for 30 min before stimulation with either natural (all trans-RA, at-RA, 5 µM) or synthetic (TTNPB, 10 µM) RAR agonists. In contrast to apo14, the other structurally related apocarotenals tested here had no effect. Veh, Vehicle. B, For concentration-dependent studies on RAR-LBD, cells were pretreated for 30 min with the concentrations of apo14 shown before stimulation with TTNPB (1 µM). C, Apo14 (30 min preincubation, 5 µM) inhibits RXR-LBD activation by its specific synthetic agonist LG100364 (LG, 300 nM). Neither apo8 nor apo12 (both 10 µM) had any RXR-LBD effect. D, Apo14, but not apo8, inhibits LBD activation of PPAR and PPAR, and to a lesser extent PPAR, by their respective ligands (listed below). Apo14 had no effect on the five other nuclear receptors shown, including the constitutively active HNF4. Cells were preincubated with apo8 or apo14 (both 10 µM, 30 min) before adding specific agonists for PPAR (WY14643, 10 µM), PPAR (carbaprostacyclin, 10 µM), PPAR (BRL49653, 1 µM), TRß (T3, 1 µM), FXR (deoxycholic acid,100 µM), LXR, 1 µM T1317 [N-(2,2,2-trifluoroethyl)-N-[4-[2,2.2-trifluoro-1-hydroxy-1-(trifluoromethyl ethyl]phenyl]-benzene-sulfonamide)], PXR (paclitaxitel, 10 µM), and VDR (1, 25-dihydrocholecalciferol, 1 µM). Responses are shown as the percent activation induced by the respective agonist alone (100%, dashed line). LBD activation (fold induction) by the respective ligands and apo14 alone is shown in the Supplemental Table. Statistical analysis was performed using Mann-Whitney test, comparing respective agonist response in the presence or absence of apo14 (*, P < 0.05).

Given these selective receptor responses, we went on to examine in greater detail those nuclear receptors most potently modulated by apo14, namely RXR, PPAR, and PPAR, testing for apo14 concentration-dependent effects, differences between its effects on synthetic vs. natural agonist responses, and direct binding of apo14 to their LBDs. Apo14 inhibited agonist-induced RXR, PPAR, and PPAR-LBD activation in a concentration-dependent manner independent of whether the respective agonist was of synthetic (Fig. 4A) or natural origin (Fig. 4B). The IC₅₀ in these cell-based assays was less than 1 µM for both PPAR and PPAR. In cell-free radioligand displacement assays, apo14 directly displaced high potency radioligands from expressed PPAR proteins in a concentration-dependent manner (Fig. 4C), although at higher concentrations (IC₅₀ for PPAR was 22.9 µM and for PPAR, 25.5 µM). Independently of the specific nature of apo14’s interaction with PPARs, these data clearly indicate that cell stimulation by apo14 but not other closely related apocarotenals inhibits adipogenesis, PPAR target gene expression, and activation of both PPAR and PPAR LBDs. These results raise the question whether apo14 treatment modulates established functional agonist-induced PPAR transcriptional responses, including trans-activation of a canonical PPAR response element direct repeat (DR1)-luciferase construct.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Apo14 Inhibits PPAR- and RXR-LBD Activation Induced by their Established Synthetic and Natural Agonists in a Concentration-Dependent Fashion LBD-GAL4 assays were repeated in HEK293 cells using PPAR-, PPAR-, or RXR-LBDs. For concentration-dependent studies, cells were pretreated for 30 min with the concentrations of apo14 shown before stimulation with either synthetic (A) or natural (B) agonists as follows: PPAR, WY(30 µM), oleic acid (18:1, 200 µM); PPAR, BRL49653 (BRL, 10 µM) stearic acid (18:0, 200 µM); RXR, LG100364 (LG, 300 nM); 9-cis RA (5 µM). C, In radioligand displacement assays (SPA), apo14 directly displaces high potency specific PPAR or - ligands in a concentration-dependent manner.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Apo14 Inhibits PPAR Response Element Activation A, A canonical DR-1 PPAR response element (acyl CoA oxidase PPRE x 3)-luciferase construct as well as ß-Gal and/or control pcDNA3 vectors were transfected into HEK293 cells before stimulation with RXR and/or PPAR agonists as specified below. Apo14 (10 µM) inhibits DR1 activation by the RXR agonist LG100364 (300 nM) either alone or in combination with known PPAR (WY, 10 µM) or PPAR (BRL, 1 µM) synthetic agonists. Veh, Vehicle. B and C, DR-1 experiments were repeated in HEK293 cells transfected with either a full-length human PPAR (B), RXR (C) or empty pcDNA3 vector (Co; B and C). In cells overexpressing PPAR, apo14 more potently inhibited DR1 activation by BRL (10 µM; B) as compared with RXR-transfected cells treated with LG (300 nM; C). After transfection, cells were pretreated for 30 min with apo14 (10 µM), followed by stimulation with the specific agonists to the respective receptor as noted. Luciferase activity was normalized to ß-Gal activity. Statistic analysis was performed using Mann-Whitney test, comparing activation by the respective agonist in the presence or absence of apo14 (#, P < 0.001; *, P < 0.05).

Apo 14 Inhibits PPAR Responses in PPAR-Dependent Manner

View larger version (38K): [in this window] [in a new window]   Fig. 6. Apo14 Inhibits Known PPAR Target Gene Responses in Vitro, in Vivo, and in a PPAR-Dependent Manner A, Apo14 inhibited mRNA expression of the PPAR target gene MCAD in thioglycolate-elicited peritoneal macrophages isolated from wild-type but not from PPAR-deficient mice (n = 10 mice/genetic background) as evident by Northern blotting. Veh, Vehicle. B, Apo14 inhibits PPAR agonist (WY)-induced mRNA expression of known PPAR target genes ACO and LCAD in a concentration-dependent manner in HepG2 cells. Cells were prestimulated with apo14 (10 µM, 3 h) followed by treatment with WY (250 µM) before Northern blot analysis was performed. C, Apo14 promotes TNF-mediated VCAM-1 expression in vitro. Apo14 increased TNF (20 ng/ml, 15 h)-induced VCAM-1 mRNA expression in human umbilical vein ECs as shown by Northern blotting; apo8 had no effect (10 µM, 3 h for both apocarotenals). D, Apo14 stimulation reversed known PPAR-mediated repression of TNF-induced VCAM-1 mRNA expression in a manner dependent on both apo14 incubation time and the order of stimulation (apo14 vs. WY). Apo14 prestimulation (10 µM) for 3 h, but not 15 min, reversed the repression of TNF-induced VCAM-1 mRNA expression in human ECs known to occur with WY (250 µM, 3 h). However, under the same concentrations/conditions as before, the opposite order of stimulation—WY first, followed by apo14 treatment—had no effect on VCAM-1 expression at either time point. E, Apo14 inhibits leukocyte recruitment in vivo in a PPAR-dependent manner. In the murine air pouch model, injection of apo14 (10 µM, 2 h pretreatment) increases the leukocyte recruitment induced by TNF (25 ng/ml for 2 h) injection into the pouch in vivo in wild-type but not PPAR-deficient mice.

Given these effects of apo14 on VCAM-1 in vitro, we next tested apo14 effects on inflammation in vivo in wild type vs. PPAR-deficient mice using the murine air pouch model, a well-established in vivo assay for leukocyte recruitment (33). After forming a sterile mature air pouch on the dorsum of mice, intra-pouch TNF stimulation was performed after prior injection of either apo14 (10 µM) or vehicle. Apo14 pretreatment increased TNF-induced leukocyte recruitment to the pouch by 600% as compared with control stimulation with vehicle/TNF (Fig. 6E). In contrast, in PPAR-deficient mice, apo14 had no effect on leukocyte recruitment. The leukocyte count induced by TNF alone was higher in PPAR-deficient mice, consistent with prior observations regarding PPAR’s role in limiting inflammation. These findings indicate that apo14 counters known PPAR responses in vitro and in vivo and in a PPAR-dependent manner. These data suggested that the presence of apo14 might be detectable in vivo under proinflammatory conditions. Although unique aspects of murine ß-carotene metabolism limit apo14 studies in mice, this is not an issue in humans. As such, we performed HPLC/mass spectroscopy on plasma from otherwise stable patients on chronic hemodialysis, a model proinflammatory condition. The plasma from hemodialysis subjects contained a unique peak with the signature characteristics of an apocarotenal and consistent with a synthesized apo14 standard (Supplemental Fig. 2). Although this peak was absent in the plasma of normal subjects (Supplemental Fig. 2), a causal role for apo14 in the clinical status of such patients cannot be concluded from these findings. Overlapping carotenoid bands also limit precise quantification of apo14 levels; comparative analysis using an apo14 standard suggests apo14 plasma levels on the order of 80 nM in the plasma of such patients.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
We provide evidence here that the asymmetric ß-carotene cleavage product apo14 represses known RXR, PPAR, and PPAR transcriptional responses induced by their respective specific agonists. These apo14 effects were evident on LBD assays, target gene expression, and well-established PPAR and PPAR functional responses in vitro and in vivo. The absence of similar responses with other apocarotenals that are quite similar in structure to apo14 combine with the receptor-specific effects of apo14 to argue for distinct, previously unrecognized transcriptional effects of this ß-carotene metabolite. To our knowledge, a natural molecule that can inhibit ligand-activation or functional effects of PPARs and RXRs either directly or indirectly has not been previously described.

The precise regulation of steroid hormone nuclear receptors is critical for metabolic homeostasis, as evident by the large cassette of genes they govern and the biologic roles of those targets (8, 34). This is particularly the case for RXR, given the number of nuclear receptors that require RXR as a heterodimeric partner (10). The specificity of these nuclear receptor responses is embedded in multiple levels, including the chemical structure of the ligand, the nature of the nuclear receptor ligand binding domain (LBD), and the cognate interaction between the two (10). The complexity of this interaction is evident in the responses reported with synthetic agonists and antagonists to PPARs and RXR. Synthetic PPAR ligands are insulin sensitizers used in type 2 diabetes to improve glucose control, whereas a synthetic PPAR antagonist can reportedly decrease weight and improve hyperglycemia in rodents (34, 35, 36). Some synthetic RXR-activating retinoids also improve metabolic parameters in animals, including body mass index, lipid profile, and glucose tolerance, whereas RXR antagonists may improve leptin resistance (8, 37). These responses appear to vary in part as a function of the specific molecule being tested (38).

Putative natural ligands for RXR and PPARs can require enzymatic action for their liberation and/or subsequent cellular access, for example as is the case with fatty acids (39, 40, 41). Similar issues are relevant to ß-carotene metabolism and the formation of retinoids that modulate nuclear receptor responses (6, 7, 8). Central enzymatic cleavage of ß-carotene in vivo generates RA at concentrations associated with RAR and RXR activation seen during vertebral development (42). Our data suggest that asymmetric ß-carotene cleavage, which leads to apocarotenal formation, may play an opposite role, generating apo14 and inhibiting RXR and PPAR responses. In cell-based assays, apo14, but not other apocarotenals, inhibited RXR, PPAR, and PPAR trans-activation by their respective known agonists. The modest effects of apo14 on RAR and LXR support previously unrecognized transcriptional effects through this ß-carotene metabolite and warrant additional study in other settings but do not likely explain the responses seen here. Apo14 suppressed PPAR and PPAR target gene expression and responses, including effects on aP2, adiponectin, and adipogenesis at low concentrations (0.1–1 µM, Fig. 2A). In cell-free radioligand displacement assays, apo14 binding to PPAR-LBDs was also evident albeit at higher concentrations (IC50 25 µM, Fig. 4C). The reason for the apo14 concentration effects in cell-free vs. cell-based assays may derive from various factors, including the generation of more potent apo14 metabolites in cell culture and in vivo and/or the presence of other proteins like retinol binding proteins that can mediate retinoid solubility, stability, and transport (7). This latter aspect of apocarotenal signaling is particularly intriguing given recent evidence for a role for retinol binding protein in diabetes (43). Importantly, modulation of nuclear receptor responses by synthetic molecules can also derive from changes in parameters such as receptor conformation, accessory molecule assembly, or heterodimer interaction as opposed to direct receptor binding characteristics (10). For example, some synthetic PPAR modulators exhibit potent biologic effects, including insulin sensitization and adipogenesis, despite weak PPAR-LBD binding (36). Although apo14 does modify LBD activation and can interact directly with PPAR-LBDs (Figs. 3 and 4), it is difficult to exclude apo14 effects on these other factors that can contribute to receptor activity, all of which will be of interest for future studies characterizing this novel pathway (10). Because both apo14 and the other apocarotenals tested here have a functional aldehyde group, this moiety is an unlikely contributor to apo14’s unique RXR and PPAR effects. Interestingly, synthetic RXR antagonists have recently been shown to preferentially stabilize RXR in an antagonistic vs. agonistic conformation through formation of a salt bridge (17). Apo14 does not appear to act exclusively through modulation of RXR given the loss of its transcriptional (MCAD and VCAM-1 expression) and functional (leukocyte recruitment) in the genetic absence of PPAR (Fig. 6). These data suggest apo14 may be able to inhibit PPAR responses independent of RXR.

Asymmetric cleavage of ß-carotene in vivo, and hence apo14 formation, has been receiving increased attention. Endogenous apo14 levels have been difficult to determine for multiple reasons, including the instability of the molecule and the inability to study endogenous apo14 in rodents due to their unique ß-carotene metabolism. The recent identification and cloning of enzymes that can specifically produce apocarotenals, like ß-carotene dioxygenase II (9'10'-monooxygenase), which yields higher carbon number apocarotenals (apo10), provides indirect support for apocarotenal generation in vivo (6). This enzyme was detected in human tissues insensitive to vitamin A deficiency, implicating this dioxygenase in biological processes other than vitamin A synthesis (44). Homogenates from several different tissues have been shown to possess enzymatic activity capable of yielding apo14 alone (45), although no specific apo14-generating enzyme has yet been reported. Various apocarotenals can be partially metabolized to apo14 during mitochondrial oxidation (23, 24, 46). Apo14 can also be formed under inflammatory or oxidative conditions, for example during spontaneous oxidation of ß-carotene or through the action of 15-lipoxygenase, potentially consistent with apo14’s proinflammatory effects (3, 47). Such formation may be consistent with our demonstrating the presence of apo14 in the plasma of patients on chronic hemodialysis. Intriguingly, the failure of ß-carotene to decrease cardiovascular disease in clinical trials, especially among smokers, also raises the possibility of connections between ß-carotene metabolites and proinflammatory responses (2, 48, 49, 50). The inhibition of nuclear receptor responses by specific apocarotenals may be worthy of further consideration as a possible molecular mechanism contributing to these ß-carotene effects.

The data presented here identify apo14 as a naturally occurring compound that can repress RXR, PPAR, and PPAR activation and suggest apo14 as a possible biologically active mediator. A biologic role for apocarotenals has remained largely obscure despite evidence that apocarotenals and enzymes that can foster their formation exist in vivo. These results also support the potential significance of distinct ß-carotene metabolites other than just RA as transcriptional modulators of cellular responses like adipogenesis and inflammation. Apo14 repression of well-established PPAR and RXR responses induced by their high potency agonists suggests apo14 studies may provide structure-function insight into how these nuclear receptors interact with modulators. The notable divergence in biologic effects between apo14 and other apocarotenals similar in structure enhance such prospects. In this regard, apo14 represents a potential molecular template for developing novel specific PPAR and/or RXR modulators. More broadly, in terms of molecular endocrinology, these apo14 data also suggest a novel regulatory model in which the cleavage of a single parent compound can lead alternatively to either the activation or inhibition of distal pathways. Although symmetric cleavage of ß-carotene and subsequent oxidation produces 9-cis RA and RXR activation, asymmetric ß-carotene cleavage can yield apo14 and inhibition of RXR and PPAR responses (Fig. 7).

View larger version (20K): [in this window] [in a new window]   Fig. 7. ß-Carotene Generates Cleavage Products that Can Direct Divergent Nuclear Receptor Responses Symmetric cleavage of ß-carotene produces two retinaldehydes that are oxidized enzymatically to the natural RXR ligand 9-cis RA. This ligand can activate RXR and thus indirectly foster the transcriptional responses of obligate RXR heterodimeric nuclear receptor partners like PPARs. In contrast, asymmetric ß-carotene cleavage produces apo14, a molecule that can inhibit RXR, PPAR, and PPAR activation by their respective agonists.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

Cell Culture
M and HepG2 cells (American Type Culture Collection, Manassas, VA) were cultured in 10% fetal calf serum/DMEM 24 h before stimulation. Human ECs from saphenous vein specimens and bovine ECs from aorta samples were isolated and cultured as before (40). Mouse fibroblast 3T3-cell line was differentiated to adipocytes and oil red O staining was performed as before (13). Briefly, cells were plated and grown for 48 h after confluence in DMEM (high glucose supplemented with 10% calf serum). Differentiation was then induced (d 0) by changing the medium to DMEM containing a standard differentiation cocktail: 10% % fetal calf serum, 3-isobutyl-1-methylxanthine (0.5 mM), dexamethasone (1 µM), and insulin (1.7 µM). Variations on stimulation protocols are as described in Results and figure legends.

Animals
PPAR ^+/+ (129S3/SvImJ) and PPAR^–/– mice were obtained from Jackson Laboratories (Bar Harbor, ME). Peritoneal M were isolated after 5% thioglycollate broth injections into 2- to 6-month-old PPAR ^+/+ and PPAR^–/– mice. The air pouch model was generated as previously described (33). Apo14 (10 µM) or vehicle was injected under sterile conditions 2 h before injection of TNF (25 ng/ml for 2 h) into the pouch in vivo in both wild-type and PPAR-deficient mice. All animals were treated in accordance with the Harvard Medical School policy on the ethical and humane treatment of animals.

Transient Transfection
Transient transfection was carried out in 24-well plates at 2.3 x 10⁴ bovine ECs/well using Fugene (Roche) (40). Human LBD-yeast Gal4 constructs were generous gifts from Drs. T. Willson (GlaxoSmithKline), B. Forman (City of Hope National Medical Center), S. Kliewer (University of Texas Southwestern), D. Mangelsdorf (University of Texas, Southwestern), P. Tontonoz (UCLA), C.-H. Lee (Harvard School of Public Health), and R. Mukherjee (Bristol Myers Squibb). PCMX-ß-galactosidase (ß-Gal) expression vector was used for transfection control. Luciferase (Pharmingen, San Diego, CA) and ß-Gal activities (Roche) were measured according to manufacturer’s protocols.

Scintillation Proximity Assay (SPA)
Radioligand displacement assays were carried out via SPA and expressed protein from human full-length cDNA for PPAR, PPAR, and PPAR₂ subcloned into the pGEX-KT expression vector (51). The ³H₂-labeled known synthetic PPAR agonists used were [³H₂]5-[4-[2-(5-metyl-2-phenyl-4-oxazolyl)-2-hydroxyethoxy]benzyl]-2,4 thiazolidinedione (nTZD), nTZD3, and nTZD4 with relative dissociation constants as follows: nTZD3, PPAR 2.5 nM, PPAR 5.0 nM; nTZD4, PPAR 1.0 nM (51).

RNA Analysis
Total cell RNA was isolated using RNeasy kit (QIAGEN, Valencia, CA), separated by 1% agarose gel, transferred to HyBond membrane (Amersham, Piscataway, NJ), and Northern blotting was performed. For probes we used mouse full coding aP2 and adiponectin cDNAs that were a kind gift from B. M. Spiegelman (Harvard University, Boston, MA) and P. Scherer (Albert Einstein College of Medicine, Bronx, NY), respectively. Human (0.75 kb) and mouse (1.5 kb) MCAD were kind gifts from D. Kelly (Washington University, St. Louis, MO) Human LCAD (0.299 kb) was from ATCC-3583156 (GenBank AI766094). L. Michalik (University of Lausanne, Lausanne, Switzerland) and W. Wahli provided rat ACO (2.0 kb), whereas human ACO (0.9 kb) was from ATCC-3443488.

Apo14 LC-MS Analysis
Plasma samples were obtained from six different randomly selected male patients (35–70 yr of age) at the University of Southern California. All subjects had been on hemodialysis for at least 1 yr and were clinically stable at the time of the blood draw (data not shown). Samples were obtained after heparin injection (as per standard protocol) and within 15 min of the onset of routine outpatient hemodialysis. Plasma (1.4 ml) was extracted by Folch method. The separation was performed gradually using a RP18 LC with photodiode array (Waters 996). Flow rate was 1 ml/min. The fractions eluted within the first 8 min were collected and analyzed using RP-18 column and isocratic separation (6% H₂O in methanol). Eluted peaks of M/Z 311 characteristic for apo14 underwent fragmentation on mass spectrometer (TOF-MS, Micromass LCT). MS was performed using electrospray ionization operating in positive ionization mode. The ionization parameters include capillary voltage, 3200 V; desolvation temperature, 300 C; source temperature, 120 C; scanning ions in the m/z ranging from 100–1000.

	ACKNOWLEDGMENTS

 
We thank D. Moller and R. Russell for helpful discussion; Drs. P. Tontonoz, B. Forman, S. Kliewer, D. Mangelsdorf, C-H. Lee, R. Mukherjee, and T. Willson for reagents; E. Schwartz, J. Qin, T. Akiama, M. Sharlach, N. Sharma, and S. Laclair for excellent technical support, and R. Tupy for editorial assistance.

	FOOTNOTES

 
Grant support included National Institutes of Health R01 HL071745, P01 HL48743, the Donald Reynolds Foundation (to J.P.); Boston Obesity Nutrition Research Center 5P30DK046200 Award, the Lerner Foundation Award, and American Heart Association SDG 0530101N (to O.Z.).

Disclosure Statement: O.Z., G.O., G.S., E.L., G.T., N.I.K., G.G.D., and J.P. have nothing to declare. J.P.B. and A.T. are employed by Merck & Co.

First Published Online September 28, 2006

Abbreviations: ACO, Acyl-CoA oxidase; apo14, ß-apo-14'-carotenal; 9-cisRA, 9-cis retinoic acid; CoA, coenzyme A; DR1, direct repeat; ECs, endothelial cells; FXR, farnesoid X receptor; ß-Gal, ß-galactosidase; HEK, human embryonic kidney; LBD, ligand binding domain; LCAD, long-chain acyl-CoA dehydrogenases; LXR, liver X receptor; MCAD, medium-chain acyl-CoA dehydrogenases; PPAR, peroxisome proliferator-activated receptor; PXR, pregnane X receptor; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; SPA, scintillation proximity assay; TR, thyroid hormone receptor; TTNPB, specific synthetic RAR ligands; VCAM-1, vascular cell adhesion molecule-1.

Received for publication May 25, 2006. Accepted for publication September 15, 2006.

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NURSA Molecule Pages Link:

Nuclear Receptors:   TRβ  |  RARα  |  RARβ  |  RARγ  |  PPARα  |  PPARδ  |  PPARγ  |  LXRβ  |  LXRα  |  FXRα  |  VDR  |  PXR  |  HNF4α  |  RXRα  |  RXRβ  |  RXRγ

Ligands:   T0901317  |  all-trans-Retinoic acid  |  Calcitriol  |  Pirinixic acid  |  9-cis-Retinoic acid  |  Thyroid hormone  |  Arotinoid acid  |  Rosiglitazone

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