Biol Cell (1993) 77, 67-76 67 ~) Elsevier, Paris

Positive regulation of the peroxisomal/3-oxidation pathway by fatty acids through activation of peroxisome

proliferator-activated receptors (PPAR)

Christine Dreyer a, Hansj6rg Keller b, Abder rah im Mahfoudi b, Vincent Laudet c, Grigorios Krey b, Walter Wahli b

aMax-Planck-lnstitut fiir Entwicklungsbiologie, A bt V fiir Zellbiologie, Spemannstr 35/V, D- 7400 Tiibingen, Germany," blnstitut de Biologie animale, Universit~ de Lausanne, B6timent de Biologic, CH-1015 Lausanne, Switzerland;

"INSERM U186/CNRS URA 1160, Institut Pasteur, 1, rue Cahnette, 59019 Lille Cedex, France (Received 3 December 1992; accepted 18 January 1993)

Summary - Peroxisome proliferators regulate the transcription of genes by activating ligand-dependent transcription factors, which, due to their structure and function, can be assigned to the superfamily of nuclear hormone receptors. Three such peroxisome proliferator- activated receptors (PPARa,/3, and y) have been cloned in Xenopus laevis. Their mRNAs are expressed differentially; xPPAR~ and /3 but not xPPARy are expressed in oocytes and embryos. In the adult, expression of xPPARa and/3 appears to be ubiquitous, and xPPARy is mainly observed in adipose tissue and kidney. Immunocytochemical analysis revealed that PPARs are nuclear proteins, and that their cytoplasmic-nuclear translocation is independent of exogenous activators. A target gene of PPARs is the gene encoding acyl-CoA oxidase (ACO), which catalyzes the rate-limiting step in the peroxisomal/3-oxidation of fatty acids. A peroxisome prolifera- tor response element (PPRE), to which PPARs bind, has been identified within the promoter of the ACO gene. Besides the known xenobiotic activators of PPARs, such as hypolipidemic drugs, natural activators have been identified. Polyunsaturated fatty acids at physiological concentrations are efficient activators of PPARs, and 5,8,11,14-eicosatetraynoic acid (ETYA), which is the alkyne homolog of arachidonic acid, is the most potent activator of xPPAR~ described to date. Taken together, our data suggest that PPARs have an important role in lipid metabolism.

peroxisome proliferator-activated receptors / fatty acids / peroxisomal/3-oxidation / gene transcription / evolution

I n t r o d u c t i o n

Fibrate hypolipidemic drugs, phtalate ester plasticizers, and trichloroacetic acid herbicides induce peroxisome proliferation in rodent liver [36], and are thus collectively called peroxisome proliferators. Although no augmenta- tion of peroxisome number could be detected in Xenopus after treatment with the hypolipidemic drug clofibrate, several peroxisomal enzymes were strongly induced, as has been observed in rodents [7]. Since this induction is at the level of transcription, it was postulated that peroxisome proliferators may regulate gene expression via ligand- activated transcription factors in a manner similar to steroid hormone receptors [51]. Recently, peroxisome proliferator-activated receptors (PPARs) have indeed been isolated f rom mouse [23], Xlaevis [11], rat [17], and man [54]. Due to their structure and function, these receptors can be assigned to the superfamily of nuclear hormone receptors which includes hormone receptors such as the thyroid hormone (TR), retinoic acid (RAR) and steroid hormone receptors [14, 60].

In this article, we review the isolation and identification of the Xenopus PPARs and describe their intracellular localization as well as their differential tissue distribution. In addition, we define the position of PPAR genes within the nuclear hormone receptor superfamily based on ami- no acid sequence comparisons. Furthermore, we describe the transcriptional control of the acyl-CoA oxidase gene by PPARs and the identification of a peroxisome prolifer-

ator response element (PPRE) within its promoter . The discovery of novel efficient activators of PPARs includ- ing natural fatty acids and the synthetic arachidonic acids analog 5,8,11,14-eicosatetraynoic acid (ETYA) is also reported.

Resu l t s and d i s c u s s i o n

Isolation and structural analysis o f PPARs from Xeno- pus laevis

We have cloned three novel members of the nuclear hor- mone receptor superfamily by screening Xenopus cDNA libraries with degenerate oligonucleotides or cDNA frag- ments encoding all or portions of the conserved DNA bind- ing domain of the nuclear hormone receptors [11]. These receptors are called peroxisome proliferator-activated receptors (PPARs) because they are stimuated by peroxi- some proliferators as described below. The three isolated Xenopus PPARs were termed ~,/3, and y in the order of their amino acid sequence similarity to the previously described mouse PPAR (fig 1A). The Xenopus PPARg may be the homolog of the mouse P P A R [23] and rat PPAR [17] because it has a high percentage of identical amino acids to these PPARs even in the usually non- conserved A and B domains, xPPAR~ more closely resem- bles the rodent PPARs than it does the/3 and y subtypes of Xenopus (fig 1A). This situation has also been observed

68 c Dreyer et al

108 173 250 474 I N B l C D l E/F xPPARct

lOl 166 244 468 I 52 I aa 79 l 87 mPPAR

101 166 244 468 I 52 ] 86 81 I 87 rPPAR

72 138 216 441 I 24 I 86 61 I 71 hPPAR/NUCI

30 95 171 396 11 1 I 78 55 l 67 xPPAR[~

112 177 257 477 I 21 I 82 43 l 64 xPPAR 7

131 198 411 593 I 24 [ 6s // I 31 ] hEAR-1

60 128 197 419 [ 14 I ; 5 7 16 t 29 I xTRuA

75 141 188 442 I ~5 I 59 17 I 25 I xRAR7

A

Fig 1. Relationship of PPARs to other nuclear hormone recep- tors. A. Amino acid sequences of nuclear hormone receptors were subdivided into domains A-F according to Krust et al [33] and aligned to the corresponding domains of xPPARa. Amino acid sequences are represented by open bars and numbers above each bar indicate the last amino acid of each domain. The highly con- served DNA-binding domain is highlighted by shading. Num- bers in the bars show the percentage of amino acid identity to the corresponding domains of xPPAR=. Sequences: xPPARs [11], mPPAR [23], rPPAR [17], hPPAR/NUCI [54], hEAR-I [40], xTR=A [6], xRART [12]. B. Phylogenetic tree connecting all members of the first subfamily of nuclear hormone recep- tors based on sequence comparisons of the central conserved por- tion of the ligand-binding domains, which comprise the Ti and

F i r s t Period

I I

Second Period

I I - - hTRcl (Human)

cTRu (C'nic~n)

- - cVERBA (Chickca)

.~xTR~A (X~opus)

~TRaB (Xcnopus)

. ~ 1 ~ BhTR~ (Human)

p (Chicken) [ ~

ARa I X e n ~ )

RI~ (Hmnan)

- - E75 (Drosophila)

m PPARa Odous¢) • 4 [ r ~

- - JtPPARtI ( X ~ )

NUCI (Human) = hPPAR ~ ?

- - xPPARp (xo'~91~a)

xPPARy (Xm~op~)

hVDR (Human)

I° • ECR (Drywall=)

B

dimerization (Ti-DM) domains according to Forman and Samuels [16]. These two domains are implicated in regulation of transacti- vation and dimerization of receptors, respectively. Sequences were aligned using the Clustal V package and the tree was calculated using the Fitch least square algorithm [34]. The shaded circles indicate the dichotomy between Drosophila and mammalian genes. The arrows point out the dichotomy between mammalian and Xenopus genes. The boxed percentages indicate amino acid differences between the mammalian and Xenopus Ti-DM sequences, which were calculated using the Clustal alignment algorithm. The bar indi- cates a length of l0 arbitrary units.

in the retinoic acid receptor subfamily, where the same sub- types from different species are more similar than the different subtypes of the same species. The closest rela- tive of the PPARs among all known nuclear hormone receptors is the putative receptor EAR-I [40]. No ligand has so far been identified for EAR-1 and thus, it is called an orphan receptor.

The P-PDox sequence of PPARs is CEGCKG. This ami- no acid sequence is located in the first Zn-finger of the DNA binding domain and it determines the specificity of DNA binding [10, 37, 59]. In analogy to other nuclear hor- mone receptors, which contain this P-Box sequence, it is predicted that PPARs recognize DNA response elements containing AGGTCA-Iike sequences [60]. A distinguish- ing feature of PPARs is the structure of the so-called D- box, which is situated in the second Zn-finger [59]. The PPAR D-box contains only three amino acids whereas in almost all other nuclear hormone receptors it contains five amino acids [34].

Evolution o f P P A R genes Evolutionary analysis of the nuclear receptor superfami- ly reveals that PPAR genes belong to the first subfamily of nuclear hormone receptors [2, 34], which comprises receptors of thyroid hormone (TR), retinoic acid (RAR), vitamin D and ecdysone, and the orphan receptors EAR-1 and E75 (fig 1B). This classification, which is based on the alignment of amino acid sequences of the Ti-DM do-

main, also illustrates the relationships between the vari- ous PPAR genes published at the present time. Clearly, there are at least three different loci : PPAR=, PPAR43 and PPARy. In the case of the recently described human PPAR, which is called NUCI [54], the situation is less clear. Comparative sequence analysis of its Ti-DM domain suggests that the isolated h P P A R might be the homolog of xPPAR~ as indicated in figure lB. The same result is obtained upon comparison of the DNA-binding domain (DBD) using the Neighbor-joining method [34]. However, analysis of the DBD by the Fitch algorithm indicates that the h P P A R is not the homolog of xPPARfl but still its closest relative. Cloning and sequence analysis of further PPAR homologs is required to determine whether this hPPAR represents a novel subtype or belongs to the fl sub- type. Since the divergence of the ,t, fl, and y PPARs pre- dates the dichotomy between mosue and Xenopus PPAR=, it is highly probable that mammalian homologs of PPAR~ and y exist. The length of the horizontal branches of the phylogenetic tree shown in figure 1B represents the diver- gence of the sequences, but it is not directly proport ional to time. Thus, a long branch could correspond to a short period of time, if the genes evolved (ie accumulated mu- tations) very rapidly. Nevertheless, it is possible to recog- nize two different periods in the evolution of this subfamily . The first period predates the a r thro- pod/ver tebrate dichotomy (approximately 500 million years before present) [32] and corresponds to the appari-

Peroxisome proliferator-activated receptors 69

0 1 4 8 9 10 11 12 15 20 24 28 39

xPPARa

xPPAR~

w N o g - , . t ° I]-actin

Fig 2. Northern blot assay of xPPARa and [3 mRNA during embryogenesis of X laevis. Total RNA from one fully grown oocyte (O), or one embryo of the indicated stage according to Nieuwkoop and Faber [43] was separated and probed with antisense RNA of xPPAR= (upper panel) or xPPARfl (middle panel). Stage 1, fertilized egg; stage 4, 8 cell embryo; stages 8 and 9, blastula; stages 10 to 12, gastrula; stages 15 to 20, neurula; stages 24 and 28, tailbud; stage 39, early tadpole. Stages 1, 8, 11, 15, 24, and 39 are depicted. Bottom panel, subsequent hybridization of the filter shown in the middle panel with a DNA-probe specific for fl-actin.

tion of the various groups of genes : TRs, RARs, PPARs, etc. The common ancestry of these receptors is supported by the recent description of a putative retinoic acid recep- tor in sponges, a group of metazoans which separated at least 540 millions years ago [4]. The second period is the diversification of receptors inside each group, eg the dupli- cation of the ancestral thyroid hormone receptor gene into two genes TRa and TR//. The three PPAR loci appeared during this second period. It is interesting to compare the speed of evolution within the TR, RAP, and PPAR groups of genes. In each group, mammalian and Xenopus homo- logs of the same genes have been described. Since the date of separation of mammalian and amphibian lineages oc- curred approximately 400 million years ago [31], the speed of divergence of the homologs after the separation of the two lineages can be estimated. We have taken the percen- tage of amino acid sequence differences in the Ti-DM do- main indicated in figure 1B to assess the divergence per 100 million years for each group. This calculation gives 1.8°70/108 years for TR (averaging the values for TR= and TR43 ), 1.1 070 for RAR (RARc¢ and RARy) and 3.40/0 for PPAR=. Although this estimation is so far only possible for PPAR=, it allows us to hypothesize that the PPAR

genes evolved approximately 2 -3- fo ld faster than TRs or RARs.

It is not known at present if the diversification events within the TR, RAR or PPAR groups occurred at the same time. Several arguments including the chromosomal loca- tion of TR and RAR genes indicate that this could be the case [34]. If this is correct, it represents a strong argument in favor of a model describing explosive diversification of nuclear receptor genes rather than de novo gene creation [25]. The dating of such a 'Big bang' of gene duplication is elusive at the present time. It is of course tempting to speculate that it corresponds to the appearance of the early vertebrates.

Expression of xPPAR¢, /3, and y in embryos and in adults

Since the cDNAs of PPAR~ and/3 were cloned f rom an ovary library and PPARy from a liver library, we have studied the expression of the different PPARs during oogenesis, embryogenesis, and in adult organs [11]. xPPARa and/3 mRNAs accumulate in early oogenesis, and a pool of maternal transcripts persists in the embryo up to early (/3) or late (=) gastrula stages (fig 2). The first

70 C Dreyer et al

Table I. Relative abundance of xPPARa,/3, and y mRNAs in organs of the adult X laevis. Northern blot analysis of xPPARa,/3, and y mRNA was performed on total (*) or polyA-selected RNA of the organs as indicated [11]. The filters were subsequently hybri- dized to a/3-actin cDNA probe. After exposure of the filters to X-ray films, the signals were quantitated by means of densitometry and each xPPAR signal was standardized to the/3-actin signal of the same lane. Arbitrary units were chosen and the strongest signals of xPPARoc,/3, and r mRNAs were defined as 100. Values for xPPARa,/3, and "r mRNA cannot be compared to each other, nd, not determined.

Oocyte Testes Liver Kidney Muscle Fat body * Brain *

xPPARa 100 12 12 54 12 8 nd xPPARfl 100 38 18 38 14 41 29 xPPARy 0.1 2 2 25 2 100 nd

zygotic transcripts of xPPARa and xPPARfl were detect- ed during or after neurulation, respectively. A gradual replacement of maternal transcripts by zygotic transcripts after midblastula transition is also seen for/3-actin, which was used as a control. No xPPARy transcripts of the ex- pected size could be detected in oocytes or embryos (data not shown). In the adult, xPPARa and/3 were expressed in all tissues tested, whereas transcription of xPPARy was predominantly observed in adipose tissue and kidney (ta- ble I). Thus, in contrast to xPPARa and/3, which appear to be ubiquitously expressed, xPPARy seems to have a res- tricted tissue distribution.

Intracellular localization o f x P P A R a and xPPAR/3

The distribution of xPPARa and/3 in Xenopus oocytes and transfected cells was analyzed by immunocytochemistry using specific antisera, xPPARa and/3 were mainly detect- ed in the nuclei of previtellogenic oocytes (fig 3A, B). In addition, xPPARfl was detected in the nuclei of follicle cells (fig 3B) and no labelling was observed with the preim- mune serum (fig 3C). The significance of the different staining patterns of xPPARa and/3 is not known, xPPARa and/3 are excluded from the nucleoli of Xenopus oocytes and transfected cells (fig 3A, D). In summary, xPPARa and/3 are predominantly localized in the cell nucleus.

Since nuclear translocation of some nuclear hormone receptors appears to be ligand-dependent (table II), we have investigated the intracellular distribution of xPPARa and/3 in transfected HeLa and cos-I cells in the presence or absence of exogenous activators. Both xPPARa and/3 were predo, xninantly detected in the nuclei of transfected cells (fig 3D, E) in the absence of exogenous PPAR acti- vators. Addition of 10 -4 M Wy-14,643, which fully acti- vates PPARs [11], did not increase the accumulation of PPARs in the nucleus (data not shown). Thus, these results indicate that PPARs are constitutively nuclear proteins. However, considering the fact that xPPARa and/3 are ac- tivated by various fatty acids (see below), we cannot ex- clude the possibility that endogenous fatty acids induce nuclear translocation. Nevertheless, we believe that this is not the case because addition of exogenous activators is required for full transcriptional activation of reporter genes by PPARs [l 1]. Alternatively, one would have to propose that nuclear translocation needs less activator than transcriptional activation, which is unlikely.

Ligand-independent nuclear translocation of PPARs is consistent with the fact that PPARs belong to the sub- group of nuclear hormone receptors whose members are all constitutively nuclear such as the TR and RAR [27]. As summarized in table II, ligand-dependent nuclear trans- location appears to be a characteristic for only a subfa-

mily of nuclear hormone receptors consisting of the glucocorticoid (GR), androgen (AR), and mineralocorti- coid receptor (MR). However, the literature is somewhat controversial on this point (see [5] and references there- in). Interestingly, a constitutive nuclear localization sig- nal (NLS) is also present in the GR, but it is repressed by the hormone binding domain (HBD) in its ligand-free state. This inhibitory property of the GR HBD can be transferred to other hormone receptors [48, 62]. For the estrogen (ER) and progesterone (PR) receptor, we have the inverse situation. Their nuclear transport becomes ligand-dependent only after deletion of the constitutive NLS [19, 62]. In the case of PPARs, sequence compari- sons did not reveal any obvious constitutive NLS similar to the one described in the GR or those postulated in other nuclear hormone receptors [57]. However, the NLS or PPARs could consist of several, dispersed, so-called proto- signals as described for the PR and ER [62]. In any case, mutagenesis studies will be required to identify the NLS of PPARs. It has been postulated that the ligand- dependent transport of steroid hormone receptors from the cytoplasm to the nucleus may be regulated by cytoplas- mic anchoring via heat shock protein 90 (hsp90) [8, 49]. Consistent with this view, the GR, AR, and MR share a hsp90 binding site in their HBD [8], whereas the TR and RAR, which are constitutively nuclear, do not bind hsp90 [9, 13, 48]. This model, however, is probably too simple since the ER and PR are constitutively localized in the nucleus although they bind to hsp90. However, binding of hsp90 to the ER appears to be different from the bind- ing to the GR in that it requires an additional site outside the HBD [53].

Peroxisome proliferators activate P P A R s

Since xPPARy was detected mainly in peroxisome proliferator-responsive tissues, we tested whether xPPARy is activated by peroxisome proliferators. For this purpose, we constructed a chimeric receptor similarly as described [14]. It consists of the DNA-binding domain of the estro- gen receptor (xER) and the ligand-binding domain of the xPPARy as indicated in figure 4. This construct, together with the appropriate reporter plasmid containing two ad- jacent estrogen response elements (ERE), was introduced into HeLa cells by co-transfection, and the activation of the reporter gene was assayed in the presence or absence of peroxisome proliferators (fig 4). The hypolipidemic drug Wy-14,643 was the most efficient activator, followed by nafenopin, and clofibrate, the anticoagulant warfarin, and the weak peroxisome proliferator trichloroacetic acid (TCA). Although the ligand-binding domains of mPPAR and xPPARy are only 61% identical in their amino acid

Peroxisome proliferalor-aclivated receplors 71

A

% -.

D

B

F

Fig 3. Nuclear localization of xPPAR,x and ,3 in oocytes and in transfected cells. Cryostat sections of X laevis ovary were stained with anti-xPPAR0c (A), anti-xPPAR/3 (B), or with preimmune serum (C) [13]. The nuclear envelope is marked with arrowheads in C and the bar represents 100 ~m and applies also to (A) and (B). Faint staining of yolk platelets in (B) and (C) is due to yellow autofluorescence which is clearly distinct from the green fluorescein secondary antibody-labelling observed on the original color pho- tographs. D. A xPPAR/3 fusion protein, which contains an influenza coat protein tag [61] was transiently expressed in cos-I cells and detected by a monoclonal antibody against the tag. Staining with a polyclonal anti-xPPAR/3 serum gave equivalent results (not shown). Heka cells were transfected with a xPPARe expression vector, fixed and stained with anti-xPPARe (E), or with the cor- responding preimmune serum (F). Cells shown in (D, E, F) were grown in the absence of exogenous activators.

72 c Dreyer et al

Table II. Cytoplasmic-nuclear transport of nuclear hormone receptors: hsp90 binding and ligand-dependency.

Receptor Length hsp90 (amino acids) binding

[8, 9,531

Ligand dependency of nuclear translocation

References

rGR 795 yes yes [47, 48, 62] hAR 919 yes yes [55] hMR 984 yes yes [I ] hPR 934 yes no [19, 20, 62] hER 595 yes no [48, 62] rTR~ 410 no no [48] xRART 442 no a no [13] xPPARct 474 ? no xPPAR/3 396 ? no

a Tested on hRARa, ~.

I 277 214 477

i L,o.oo i I I I

xER xPPARy

lx10 "S SxlO "S lX10 "4

lx10 "S SxlO "5 lx10 "4

=E lx10"5

",~ i X10-4 SxlO'4 lx10"3

g u

fold induction

SO 100 150

Wyq 4,643

L

Nafenopin

Clofibrate

H N ' ~ S vCOOH

Cl

~_o~COO. C l - - ~ O COOH

lX10.4 ~ Warfarin

2x10 °4 5XI0 "4

0

2xi0"3 I I I Trichloroacetic acid 5x10-3 ~ CI COOH lxlO'Z CI CI

Fig 4. Activation of the xPPARy ligand-binding domain in a chi- meric receptor. HeLa cells were co-transfected with a plasmid encoding the chimeric receptor xER-xPPAR T, whose schematic structure is shown on top, and a CAT reporter plasmid contain- ing two adjacent ERE consensus sequences. CAT activities were normalized to luciferase activity obtained by co-transfection of the luciferase expression vector. The fold inductions observed with the xenobiotic peroxisome proliferators Wy-14,693, nafenopin, clofibrate, warfarin and TCA at the concentrations indicated are shown. For absolute CAT activities, see [11]. Ex- periments were done in triplicate and the mean values together with the standard deviations are shown. The chemical structures of the activators are shown on the right.

sequence, we found the activator-specificity for xPPAR T to be identical to that of the mouse P P A R [23]. Further- more, Wy-14,643 was also found to be the most potent activator of xPPAR~t and /3 among the peroxisome proliferators mentioned above (data not shown). Maximal

stimulation of PPARs required activator concentrations of 10 -4 M or greater, depending on the activator. This is at least three orders of magnitude above the concentra- tions at which steroid hormones or retinoids fully activate their nuclear receptors [14, 22, 38], which indicates that peroxisome proliferators may not be high-affinity ligands of PPARs. Alternatively, peroxisome proliferators may not get efficiently transported into cells or may be quick- ly degraded so that their actual effective intracellular con- centrations are very low.

Characterization of a PPRE in the 5' upstream region of the acyI-CoA oxidase gene

The structure of the P-box, which is composed of the se- quence CEGCKG in all PPARs, suggests that PPARs bind to response elements whose half site consensus sequence is AGGTCA [60]. Several AGGTCA-like sequences are present in the 5' upstream region of the gene encoding acyl- CoA oxidase (ACO) [46]. This enzyme, which catalyzes the rate-limiting step of the peroxisomal/3-oxidation path- way of fatty acids [35], is induced by peroxisome prolifer- ators [52]. Thus, we and others [58] have tested whether the rat ACO gene is regulated by PPARs. Cotransfection experiments utilizing PPAR expression vectors and differ- ent CAT reporter plasmids containing the rat ACO gene promoter or parts of it were performed and the results are summarized in figure 5. Presence of the 5' flanking region from - 1273 to + 20 of the ACO gene led to induction of the reporter gene in the presence of Wy-14,643 by all three xPPARs, but not by the vector pSG5 alone. To local- ize the peroxisome proliferator response element (PPRE) within the ACO promoter, we first tested a truncated promoter consisting of nucleotides - 1273 to - 4 7 5 and subsequently, two subregions ACO-A ( - 578 to - 553) and ACO-B ( - 543 to - 520). ACO-A and ACO-B have been previously identified as regulatory sequences in the perox- isome proliferator responsive enhancer region of the ACO gene [46]. As shown in figure 5, the truncated promoter and the ACO-A sequence, but not ACO-B, mediate trans- activation by PPARs. Therefore, ACO-A represents the PPRE within the ACO gene promoter. Although report- er plasmid inductions were highest with xPPAR~ (table III), absolute CAT activities were 3-8- fo ld higher with xPPARcx compared to xPPAR/3 or y. Moreover, a 3-15-fold activator-independent stimulation of PPRE- containing plasmids was observed in the presence of PPARa¢ [11, 27]. This could be due to low amounts of en-

Peroxisome proliferator-activated receptors 73

dogenous activators, or to a significant constitutive PPAR~ activity. As predicted, ACO-A contains several AGGTCA-I ike sequences which are putative PPAR bind- ing sites. Recently, we and others have shown by gel retardation experiments that PPAR bind to this PPRE in the form of heterodimers with the retinoid X receptor (RXR) [26, 27, 301.

fo~ i n d t ~ t l o n

ACO-A ACO-B ,/ xPPARct xPPARI~ xPPARy pSGS

. I z r 3 . zo

,,co.c,,, - t I CAT J--- 3 ,3 4

• 12 r3 .4VS

ACO.G.C~T "~1 IIR IG I CAT ~-- 4 10 6 Z

• STS "SS)

• S43 -5ZO

ACO-B.TK CAT [~1 . . . . . . . . . . . ~ . . . . . . . . . . . ~ 1 1 ] I

Fig 5. Identification of the PPAR regulatory element in the acyl- CoA oxidase gene promoter. CAT reporter plasmids containing the full length acyI-CoA oxidase gene promoter ACO.CAT ( - 1273/+ 20), a truncated version ACO.G.CAT ( - 1273/- 475) in front of the heterologous rabbit fl-globin (G) basal promoter, or the short sequences ACO-A ( - 578/ - 553) or ACO-B ( - 543/ - 520) in front of the herpes simplex virus thymidine kinase (TK) promoter were co-transfected with xPPAR~,/3, "r or the expression vector pSG5 into HeLa cells [1 !]. Following induction with 10 -4 M Wy-14,643 or without induc- tion as a control, CAT activities were measured in cell extracts and the means of the fold inductions are tabulated. Perfect and imperfect AGGTCA motifs, which are putative PPAR binding sites, are indicated by solid and shaded arrows, respectively.

Table Ill. Activation of xPPARa by fatty acids. The xPPAR~ expression vector and the ACO-A.TK.CAT reporter plasmid were cotransfected into HeLa cells. Subsequently, fatty acids or sol- vent as a control were added to the cell culture medium and CAT activities were assayed. 100% CAT activity was taken arbitrari- ly as the CAT activity observed with 50 ~M arachidonic acid. Experiments were done at least in triplicate and the mean values are shown.

Each 50 t~M % CA T activity

Non-induced 21 Wy-14,643 99 3,3',5 Triiodothyroacetic acid 18

Polyunsaturated fatty acids Docosahexaenoic acid C22:6w3 146 Eicosapentaenoic acid C20:5w3 92 Linolenic acid CI 8:3w3 102 Linoleic acid C18:2w6 106 Arachidonic acid C20:4~6 100

Monounsaturated fatty acids Petroselinic acid C 18: lwl 2 94 Oleic acid C 18: I to9 60 Elaidic acid C 18: I w9 trans 59 Erucic acid C22:lw9 16 Nervonic acid C24:lto9 14

Saturated fatty acids Lauric acid C12 45 1,12 Dodecanedioic acid C12 24

A putative PPRE, with a sequence very similar to the ACO PPRE, has also been found in the 5' flanking region of the gene encoding the bifunctional enzyme (enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase), which catalyzes the second and third reactions of the peroxisomal /3-oxidation pathway [63]. Furthermore, the CYP4A6 gene, which encodes a member of the peroxisome-proliferator induced cytochrome P450 fatty acid w-hydroxylase fami- ly [28], is activated by PPARs through a PPRE similar to the ACO-A PPRE [41]. Cytochrome P450 enzymes are well known for their role in drug-catabolism, but they cata- lyze also important reactions in the metabolism of en- dogenous lipophilic compounds such as steroid hormones, fatty acids or cholesterol [42]. It will be interesting to de- termine whether other P450 genes are regulated by PPARs.

Fatty acids activate P P A R s

Structural similarities between peroxisome proliferators and fatty acids, in addition to reports that long-chain fatty acids induce peroxisome proliferation in yeast [29] and peroxisomai/3-oxidation in rats [3], led us to investigate whether fatty acids activate PPARs. HeLa cells were cotransfected with the xPPAR~ expression vector and the ACO-A.TK. CAT reporter plasmid and various fatty acids were subsequently added to the culture medium. CAT as- says revealed that polyunsaturated fatty acids (PUFAs) ac- tivated xPPAR~ as efficiently as did Wy-14,643 (table III). The inductions were 4 -8 - fo ld and there was no signifi- cant difference between w-3 and w-6 PUFAs. Mono- unsaturated fatty acids (MUFA) gave more heterogene- ous results; whereas petroselinic acid was as active as the PUFAs, the very-long-chain fatty acids erucic acid and ner- vonic acid did not activate xPPARg. In this context, it should be mentioned that very-long-chain fatty acids are exclusively metabolized in peroxisomes [24, 45], and that they exert an inhibitory effect on the peroxisomal /3- oxidation in vivo [15]. The saturated fatty acid lauric acid and the dicarboxylic acid dodecanedioic acid were poor activators (table III). Similar results have been obtained for xPPAR~ and y (data not shown). Taken together, these results indicate that physiological concentrations (50 tzM) of PUFAs activate PPARs as efficiently as does the po- tent peroxisome proliferator Wy-14,643.

PUFAs are the precursors of important cellular signall- ing molecules such as the prostaglandins, leukotrienes and lipoxins. Thus, we investigated whether arachidonic acid itself or known metabolites of it might activate PPARs. Common inhibitors of the cyclooxygenase, lipooxygenase, and epoxygenase pathways [50], which are the known arachidonate pathways for the generation of the signall- ing molecules mentioned above, were tested for their ability to repress the activation of xPPAR~ by arachidonic acid. Neither aspirin (100 ~zM) nor indomethacin (10 ~zM) which inhibit the cyclooxygenase pathway, or nordi- hydroguaiaretic acid (10/~M), an inhibitor of the lipo- oxygenase pathway, or metyrapone (10/zM) which inhibits the epoxygenase pathway, impaired the activation of xPPAR~ by 10 ~zM arachidonic acid (data not shown). Consistent with these results, the prostaglandins PGD z, PGE 2, and PGFz~ (10 tzM each) and the hydroperoxy- eicosatetraenoic acids 5-, 8-, 12-, and 15-HPETE (0.6 tzM each) did not activate xPPARa (data not shown).

Surprisingly, the synthetic arachidonic acid-homolog 5, 8,11,14-eicosatetraynoic acid (ETYA) fully activated xPPARa at a concentration of only 10 -6 M. ETYA is an inhibitor of the lipooxygenase and cyclooxygenase by act-

74 C Dreyer et al

>

I-- <

o>

2OO

100

H S N N v C O O H

Cl Wy-14.64a

10 "10 10 .9 10 .8 10 "7 10 "6 10 "s 10 4 10 3 10 "~

log (M)

Fig 6. Activation of xPPAR=. Dose-response curves of 5,8,11,14-eicosatetraynoic acid (ETYA), arachidonic acid and Wy-14,643. °70 CAT activities were determined as described in table III. 100070 CAT activity was chosen arbitrarily as the CAT activity observed with 50 #M arachidonic acid. Higher concen- trations of activators than those shown were cytotoxic or led to detachment of the cells. Mean values of at least three indepen- dent experiments are shown.

ing as a false substrate [56]. Based on the corresponding EDs0 values obtained f rom dose response curves, xPPAR~ is activated 100 times more efficiently by ETYA than by Wy-14,643 or arachidonic acid (fig 6). In conclu- sion, natural as well as synthetic fatty acids activate PPARs as efficiently as do xenobiotic peroxisome proliferators [26, 271.

PPARs are the first example of eukaryotic transcrip- tion factors which are activated by nutrients (ie fatty acids) and which regulate the expression of enzymes involved in the metabolism of these nutrients. Of course, this resem- bles very much the positive gene control by environmen- tal compounds found in prokaryotes such as the arabinose metabolism in E coli. Thus, it is tempting to speculate that PPARs may represent primitive members of the nuclear hormone receptor superfamily or of the eukaryotic tran- scription factors in general, whose existence has been postulated [44]. Although direct binding of fatty acids to PPARs or alternatively, the structure of the true ligand of PPARs remain to be demonstrated, the fact that PPARs are activate41 by fatty acids and xenobiotics and that PPARs regulate the metabolism of these latter substrates is of great physiological importance.

Hormonal control o f the peroxisomal ~-oxidation path way

It is well known that xenobiotics including fibrate hypolipi- demic drugs, phthalate ester plasticizers and synthetic fatty acid derivatives as well as natural fatty acids are able to induce the enzymes of the peroxisomal/3-oxidation path- way at the transcriptional level [45]. It is now clear that this induction is mediated by ligand-activated transcrip- tion factors called PPARs [11, 23, 26]. In addition to the inducers mentioned above, dehydroepiandrosterone (DHEA), dexamethasone (DEX) and conditions such as hyperthyroidism and vitamin E deficiency have been reported to cause induction of hepatic peroxisomal /3- oxidation [45]. Presently, it is not known whether DHEA and DEX or the agents involved in the aforementioned conditions act via their own ligand-activated transcription factors or whether they work through PPARs. An action

on PPARs, however, would necessarily be an indirect one since DHEA, DEX, tri-iodo-thyronin and ~-tocopherol have no effect on PPARs in transfection assays [11, 23]. Recently, it has been reported that the peroxisomal /3- oxidation genes are also induced by retinoic acid imply- ing a role for retinoid receptors [21]. We and others have analyzed the convergence of the retinoid and PPAR sig- nalling pathway and found that the two pathways cooper- atively stimulate the acyl-CoA oxidase gene promoter and that they interact directly in the form of P P A R / R X R het- erodimers which bind cooperatively to the identified PPRE [26, 30]. It is conceivable that PPARs heterodimerize with further transcription factors mediating the induction of the/3-oxidation system by the other substances or condi- tions mentioned previously. Alternatively, these substances could activate independent signalling pathways working through different response elements in the gene promoters.

Apart from the tissue distribution, we have so far not been able to demonstrate any qualitative or quantitative differences in the functioning of the PPAR subtypes. However, closer examination with respect to transactiva- tion of different target genes, heterodimerization with other transcription factors or activator specificity of new inducers may reveal physiologically significant differences. In addition, the generation of transgenic mice or the knock-out of PPAR genes by homologous recombination should yield answers to this question. Finally, the obser- vation that xPPARg and/3 are apparently ubiquitously ex- pressed and that PPARs control a fundamenta l biochemical process in lipid metabolism suggests that PPARs fulfil essential functions during development and in adulthood.

Acknowledgments

The expert technical assistance of F Givel, M Perroud and B Gl~iser is gratefully acknowledged. We wish to thank D Stehelin for stimulating discussions and S Child for critical reading of the manuscript. The work was supported by the Swiss National Science Foundation, the Etat de Vaud, and the Deutsche For- schungsgemeinschaft.

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