Institut de Biologie Animale (G.K., O.B., M.P., W.W.),
Bâtiment de Biologie, Université de Lausanne, CH-1015
Lausanne, Switzerland,
Molecular Endocrinology Laboratory
(F.L., E.K., M.G.P.), Imperial Cancer Research Fund, 44
Lincolns Inn Fields, London WC2A 3PX, U.K.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent work has shown that antidiabetic compounds of the
thiazolidinedione class and a prostaglandin J2 metabolite
are ligands of PPAR (9, 10, 11), whereas the experimental hypolipidemic
drug Wy 14,643 and the natural inflammation mediator leukotriene
B4 (LTB4) are PPAR
ligands (12). These findings raise
the intriguing possibility that a broad range of natural and synthetic
molecules can interact directly with PPARs to regulate target gene
expression. To test this hypothesis, we had to overcome one of the
limitations of the classic binding assay, which is the availability of
radiolabeled candidate ligands. To that end, we developed a novel
in vitro ligand detector assay that we have termed
Coactivator-dependent receptor ligand assay (CARLA). CARLA is based on
the recently reported ligand-induced binding of transcriptional
mediators, such as RIP 140 (13), TIF1 (14, 15), TIF2 (16), SRC-1 (17),
SUG1 (15), and CBP (18), to classic nuclear receptors. Whereas SRC-1
and TIF2 function as transcriptional coactivators, the role of the
other proteins remains unknown. Herein, we have used SRC-1, which
promotes the transcriptional activity of the progesterone,
glucocorticoid, estrogen, thyroid hormone, and retinoic-X receptors,
through direct interaction with the ligand-activated form of the
receptors (17). Using CARLA, we demonstrate that various naturally
occurring fatty acids and metabolites, as well as synthetic
hypolipidemic compounds, by virtue of their ability to induce
PPAR/SRC-1 interactions, are bona fide PPAR ligands. The
identification of fatty acids and eicosanoids as ligands of nuclear
receptors implies that these compounds can regulate gene expression
through the same mechanism of action used by steroid hormones.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For the validation of the interaction assay we determined whether the
known PPAR ligands can induce interactions of the receptor with SRC-1.
From the four PPAR ligands identified by classic receptor-ligand
binding assays, BRL 49653 and 15d-12,14-PGJ2
were identified using the LBD of the mouse (m) PPAR
(9, 10, 11) whereas
Wy 14,643 and LTB4 were shown to bind to the Xenopus PPAR
LBD (12). Neither our group nor others (S. Kliewer, personal
communication) was successful in obtaining soluble GST-PPAR LBD fusion
protein when the LBDs were of mammalian origin, i.e. mouse
or rat. In contrast, the LBDs of the three Xenopus PPAR
subtypes, xPPAR
, ß and
, could be purified as
glutathione-S-transferase (GST)-fusion proteins from
Escherichia coli extracts. Because the dissociation
constants (Kds) of both PPAR
ligands, BRL 49653 and
15d-
12,14-PGJ2, have not been determined
with the Xenopus receptor and because we have observed
species-specific differences in the activation of PPARs by their
activators/ligands (see below and H. Keller and W. Wahli, unpublished
results), relatively high concentrations (100 µM) of the
PPAR ligands were used in the initial experiments. As shown in Fig. 1
, the four known PPAR ligands induced binding of
in vitro translated and 35S-radiolabeled SRC-1
to purified GST-xPPAR
LBD and xPPAR
LBD fusion proteins
immobilized on glutathione Sepharose beads. These interactions were
specific both to the PPAR LBDs and to SRC-1. Thus, the PPAR
ligands
induced specific SRC-1/PPAR
-LBD interactions as was also the case
for the PPAR
ligands that induced only PPAR
-LBD/SRC-1
interactions (see Fig. 2
). In addition, there was no
induced binding of SRC-1 by the PPAR LBDs when ligands such as
17ß-estradiol and 9-cis-retinoic acid were used.
Similarly, PPAR ligands did not induce interactions between the PPAR
LBDs and in vitro translated estrogen receptor (not shown),
implying that these compounds do not enhance nonspecific
protein-protein interactions, or chemically cross-link proteins.
Finally, GST alone did not retain significant amounts of SRC-1 in the
presence of ligands (not shown). In preliminary experiments, deletion
of the activation function 2 core of PPAR
suppressed induced binding
of SRC-1 (G. Krey and W. Wahli, unpublished). Taken together, the above
results indicate that the ligand-dependent PPAR/SRC-1 interaction
assay, termed coactivator-dependent receptor ligand assay or CARLA,
provides a new method for the identification of PPAR ligands. In the
following sections, we describe the use of CARLA for the identification
of bona fide PPAR ligands, among the known activators of the
three PPAR subtypes from Xenopus.
|
|
|
Arachidonic Acid Metabolites Are PPAR Ligands
The essential fatty acids, through arachidonate, are the
precursors of the prostaglandin, thromboxane, and leukotriene families
of compounds. These compounds, collectively known as eicosanoids, have
important and diverse biological effects ranging from controlling
platelet aggregation to chemotaxis. A number of eicosanoids have been
shown to activate PPAR in cellular transfection assays (10, 11, 12, 20, 21). Furthermore, two of these compounds, LTB4 and
15d-
12,14-PGJ2, are ligands for the PPAR
and
-subtypes, respectively (10, 11, 12). As discussed above, these
results have also been confirmed by CARLA (Figs. 1
and 2
). Another
arachidonic acid metabolite, which has been shown to be a strong
activator of the human (h) PPAR
, is the lipoxygenation product
8(S) hydroxyeicosatetraenoic acid \[8(S)-HETE\].
Interestingly, 8(S)-HETE stereoselectively activates the
receptor over its 8(R)-enantiomer (20). As shown in Fig. 2
, when 8(S)-HETE is tested by CARLA it induces strong
xPPAR
-SRC-1 interactions. Similarly, 8(R)-HETE is also a
xPPAR
ligand, albeit of much lower affinity than its
8(S)-enantiomer. Indeed, dose-response curves reveal CARLA
ED50s of 1 and 50 µM for 8(S)-HETE
and 8(R)-HETE, respectively (Fig. 3A
), indicating strong
stereoselectivity in ligand binding. 8(S)-HETE is also a
ligand of xPPARß and
with CARLA ED50 values of 20 and
>50 µM for the two receptors, respectively (Figs. 2
and 3B
and results not shown), whereas no interaction was detected between
8(R)-HETE and the ß- and
-receptors (Fig. 2
). In
accordance with results of cellular transfection assays in which the
chemotactic agent 5(S)-HETE fails to activate PPARs (20),
this compound does not induce interactions of SRC-1 with either of the
xPPAR subtypes (Fig. 2
) and serves as an additional control for the
validity of CARLA.
In contrast to 8(S)-HETE, which can induce
xPPAR-SRC-1 interactions at low concentrations, LTB4, an
xPPAR
ligand with a Kd of 0.1 µM as
determined by Scatchard analysis (12), is required at 100
µM concentration to induce maximum xPPAR
-SRC-1
interactions. In addition, even at this concentration, the amount of
SRC-1 retained by the PPAR
LBD is significantly lower compared with
that retained due to 8(S)-HETE, or the fatty acids (
30%,
Fig. 2
). However, as illustrated in Fig. 3A
, a reproducible biphasic
CARLA behavior is observed with LTB4. Thus, the CARLA value due to 0.1
µM of this compound is
40% of the value seen with 100
µM LTB4. This value then remains constant up to
concentrations
10 µM, at which point the amount
of retained SRC-1 is increased. The reason and significance of the
unique CARLA behavior of this PPAR ligand remain to be elucidated.
In conclusion, the above results demonstrate that 8(S)-HETE
with a ED50 of 1 µM for xPPAR (Fig. 3A
) is
a potent natural ligand of this subtype. Note that xPPAR
has a
50-fold higher affinity for 8(S)-HETE than for its
precursor, arachidonic acid (CARLA ED50
50
µM, not shown). 8(S)-HETE, linolenic, and
linoleic acids with ED50s of
20 µM (Fig. 3B
) are presently the best natural ligands for xPPARß. For
xPPAR
, 15d-
12,14-PGJ2 with a CARLA
ED50 value of 20 µM (Fig. 3C
) is the most
potent natural ligand identified so far for this subtype, as is also
true for mPPAR
(10, 11).
Hypolipidemic Drugs Are Ligands of xPPAR, -ß, and -
PPAR activators also include synthetic hypolipidemic agents of
experimental interest or that are already being used for the treatment
of hyperlipidemia in humans. However, the molecular mechanism(s) of
action of these drugs remain unclear. As a first step in analyzing
these mechanisms with respect to PPAR biology, we determined whether
hypolipidemic drugs directly bind to PPARs and whether they do so in a
receptor subtype-specific manner. In addition to Wy 14,643 and BRL
49653, used above to validate the CARLA assay (Fig. 1B), we tested
5,8,11,14-eicosatetraynoic acid (ETYA), a synthetic arachidonic acid
analog and potent xPPAR
activator (7), the medically important
bezafibrate, clofibrate, ciprofibrate, fenofibrate, and nafenopin (also
a fibrate), as well as the nonfibrate hypolipidemic drugs, benfluorex,
gemfibrozil, and probucol, for their ability to induce interaction of
SRC-1 with xPPAR
, -ß, and -
. As shown in Fig. 2
, each of the
PPARs interacted with two or more of the above mentioned compounds,
i.e. PPAR
with ETYA and Wy 14,643, PPARß with ETYA and
bezafibrate, and PPAR
with BRL 49653 and ciprofibrate. In addition,
partial overlap in ligand recognition between the receptor subtypes was
observed. This is most evident for ETYA with ED50s of 1 and
50 µM for PPAR
and -ß, respectively (Fig. 3
, A and
B), and with only low affinity for xPPAR
(Fig. 2
). In contrast, some
compounds exhibit a more strict subtype specificity, such as BRL 49653
for xPPAR
with an ED50 of 8 µM (Fig. 2
and 3C
) and Wy 14,643, which acts preferentially on xPPAR
(Fig. 2
).
Among the fibrate hypolipidemic agents, the most potent, with respect
to PPAR subtypes, are Wy 14,643, with an ED50 of 30
µM, bezafibrate, with an ED50 of 5
µM, and ciprofibrate, with an ED50 of 30
µM for xPPAR
, -ß and -
, respectively (Fig. 3
).
The nonfibrate drugs, benfluorex, gemfibrozil, and probucol, do not
interact with either PPAR subtype at the concentration tested (100
µM). These data provide evidence that the fibrate
hypolipidemic agents are PPAR ligands to which they interact with
subtype-specific preference. Consequently, the biological action of the
hypolipidemic PPAR ligands could be mediated by one or all of the
receptor subtypes, depending upon which ligand is being used. It is
also interesting to note that the ED50 values of 1 and 30
µM for ETYA and Wy 14,643, respectively, in this
interaction assay (Fig. 3A
) correlate well with the ED50s
of 0.8 and 2030 µM for these compounds in a cellular
transcriptional activation assay (7). Furthermore, the relatively poor
xPPAR
activator clofibrate, with an ED50 of 200
µM in transfection assays (H. Keller and W. Wahli,
unpublished), is required at concentrations of 100 to 1000
µM to induce interaction between PPAR
and SRC-1 (not
shown).
In conclusion, the fatty acid analog ETYA, bezafibrate, and the
thiazolidinedione antidiabetic drug BRL 49653 are the most potent
synthetic ligands identified so far for the xPPAR, -ß, and -
subtypes, respectively. Our results also indicate that PPAR ligand
recognition has been preserved through evolution as exemplified by the
binding of BRL 49653 to PPAR
of lower vertebrates (this study) and
of mammals (9, 11).
PPAR-Ligand Interaction and Transcriptional Activation
The CARLA profiles (dose-response curves) obtained with the fatty
acids, as well as with Wy 14,643 and ETYA (Figs. 1 and 3
), correlate
very well with the transcriptional activation profiles of the
Xenopus PPAR receptors by these compounds (7, 19). However,
several of the ligands studied herein by CARLA, i.e.
8(S)- and 8(R)-HETEs, bezafibrate, BRL 49653, and
15d-
12,14-PGJ2, have not been previously
tested for their ability to transcriptionally activate the PPARs of
Xenopus. Thus, we assessed whether the dose-dependent
PPAR-SRC-1 interactions observed in vitro with these
compounds parallel their effect on transcription in transfected cells.
Because PPAR ligand binding can exhibit strong species-specific
differences (H. Keller and W. Wahli, unpublished results), we examined
in parallel the transcriptional activation profiles of the mouse and
human PPAR
in response to 8(S)- and 8(R)-HETE
and of the mouse and human PPAR
in response to BRL 49653 and
15d-
12,14-PGJ2. The Xenopus
receptors present an excellent correlation between the
ED50s obtained in CARLA and in transfection experiments
(Fig. 4
). For xPPAR
and its ligand
8(S)-HETE for example, the transcriptional activation
ED50 is 2 µM compared with 1 µM
in CARLA. Also consistent with the CARLA results, 8(S)-HETE
is a much more efficient activator of PPAR-dependent transcription than
its 8(R) stereoisomer. Furthermore, 8(S)-HETE,
which did not induce xPPARß/SRC-1 interactions at low concentrations,
also failed to significantly activate transcription through the
ß-receptor (compare corresponding panels in Figs. 3
and 4
). Finally,
species comparison indicates that the mPPAR
has a dose response to
8(S)- and 8(R)-HETEs similar to that of its
Xenopus homolog, while the hPPAR
responds slightly better
to both of these compounds (Fig. 4
).
|
Together, the above results demonstrate that the Xenopus PPAR ligands identified herein by CARLA are also activators, i.e. agonists, of PPAR-dependent transcription and that the ligand-receptor and transcriptional activation dose-response curves are in agreement. In addition, they show that the corresponding PPAR subtypes from different species respond similarly to ligands in terms of specificity and stimulation of transcription.
Compounds Negative in CARLA and Positive in Transactivation
All compounds, so far without exception, that induce PPAR-SRC-1
interaction are active in mediating transcriptional stimulation.
Conversely, one could expect that compounds that fail to induce
PPAR-SRC-1 interactions in CARLA are also unable to activate
PPAR-dependent transcription. Indeed, this is the case with the
nonfibrate hypolipidemic drugs, benfluorex, gemfibrozil, and probucol,
as well as with the nervonic and 1,12-dodecanedioic acids, or
5(S) HETE that are negative both in CARLA (herein) and in
transactivation (Ref. 7 and results not shown). In contrast to the
above compounds, fibrates such as bezafibrate, ciprofibrate, and
nafenopin significantly activate all three PPAR subtypes in HeLa cells,
albeit to different levels (Fig. 5). At the same
concentration (100 µM), fibrates either fail to induce
strong PPAR-SRC-1 interactions in vitro or are
subtype-specific ligands (compare Figs. 2
and 5
). Of these compounds,
nafenopin appears to stimulate transcription 8- to 15-fold through all
three xPPAR subtypes, which confirms previous observations in similar
experiments (5). Also of interest is the observation that BRL 49653
confers some activation to xPPAR
and, to a lesser extent, to
xPPARß, when present at micromolar concentrations, although this
compound is not defined as xPPAR
or -ß ligand by CARLA (Fig. 2
).
Similarly, it also activates mPPAR
and hPPAR
(Fig. 5B
) although
it was defined as a mPPAR
-specific ligand (9, 11). Activation of
PPARs by compounds that are not identified as ligands in two different
assays, i.e. CARLA and classic ligand-binding assay,
suggests that such compounds can indirectly influence PPAR-dependent
transcription possibly as metabolites or by releasing endogenous
ligands. Alternatively, it is possible that these compounds are indeed
ligands but that they confer a distinct structural conformation to the
receptor that excludes PPAR/SRC-1 interactions without, however,
excluding interactions with other coactivators.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Compared with the classic ligand-binding assay, CARLA presents major technical advantages: it does not require radioactive labeling of candidate ligands, it is sensitive and, in addition, identifies only agonist ligands. The technical simplicity of the assay makes possible the screening of a large number of compounds with simultaneous economic advantages in terms of material and time. In addition, the fact that this assay is based on a physical-functional interaction between two proteins in response to ligand, and not merely on the association of a lipophilic molecule with a globular protein domain, suggests that it should be less prone to artifacts. However, the assay depends on soluble GST-receptor fusion protein produced in bacteria, which can not always be taken for granted (see Results). In addition, some compounds, which might induce conformational changes that may result in interactions with coactivators different from SRC-1, would escape identification as ligands when SRC-1 is used in the assay system. Finally, but not least, it should be noted that the CARLA screen should be applicable to the identification of ligands for other orphan members of the nuclear hormone receptor superfamily.
Our results also indicate that there is an excellent correlation between the CARLA ED50 values and transactivation ED50s observed in transient transfection experiments. However, further work, beyond the scope of the present study, is necessary to establish how and if the CARLA ED50 values relate to affinity constants of the receptors for their ligands.
PPARs: Promiscuous Receptors with Overlapping Ligand
Recognition
We have demonstrated that the three PPAR subtypes from
Xenopus are able to bind a wide spectrum of compounds
ranging from fatty acids and arachidonate metabolites and analogs to
synthetic hypolipidemic drugs. The amazing ability of the PPAR LBDs to
accommodate such a variety of molecular structures implies that these
receptors, in contrast to the steroid/thyroid hormone receptors with
their stringent ligand specificity, have evolved under different
selection pressures. In addition, there is an important overlap in
ligand recognition between the three PPAR subtypes, especially for
polyunsaturated fatty acids. Overlap in ligand recognition does not
necessarily imply functional similarities between PPAR, -ß, and
-
, since differences in the affinities of each subtype for the
ligands, as well as the tissue distribution of the subtypes, may be
important determinants of subtype- and tissue-specific recognition and
regulation of PPAR target genes.
Our data also indicate that, in general, ligand recognition by PPAR
subtypes has been preserved through evolution. However, together with
results from others (9, 11), they also show that species-specific
differences exist in the affinity of a receptor subtype for a given
ligand, as is suggested by the fact that hPPAR is more efficiently
activated by 8(S) HETE than either the mouse or
Xenopus receptors, whereas the mouse and human PPAR
are
more efficiently activated by BRL 49653 than their Xenopus
homolog. It remains to be investigated whether species-specific
differences in affinity for natural ligands reflect
species-specific physiological peculiarities, such as peroxisomal
proliferation.
Fatty Acids as Hormones
In mammals, synthesis of the 6 and
3 series of fatty acids
depends on the nutritional intake of the essential fatty acids,
linoleic, linolenic, and arachidonic acids. Production of many
biologically important molecules, such as prostaglandins, thromboxanes,
and leukotrienes, depends on the intracellular availability and levels
of the essential fatty acids and their derivatives. Our identification
of unsaturated fatty acids as PPAR ligands provides the most conclusive
evidence to date that part or all of PPAR-dependent transcription
(reviewed in 3 results from the direct activation of the receptor
by these molecules and further implicates PPARs as key control factors
in the maintenance of energy homeostasis. Consequently, fatty acids
that share properties of classic hormones, i.e. the ability
to directly interact with nuclear receptors and to regulate
transcription, can no longer be considered as simple biological
substrates. Even though the actual concentration of fatty acids in the
cell nucleus, where PPARs are localized, has not been extensively
documented, the levels of free (nonesterified) fatty acids in human
blood plasma (22) are sufficiently high for PPAR activation. This
implies that high-affinity binding of fatty acids to PPARs would not be
an advantage as these receptors have evolved to sense ligands whose
concentrations are naturally high and even very high in situations of
lipid overload.
Although the PPAR subtypes overlap in ligand recognition, there is a
clear preference of the -subtype for PUFAs. It remains still to be
demonstrated whether this preference is linked to the adipolytic
function of PPAR
(review in 3 . Similarly, the strong
preference of the monounsaturated fatty acids for PPAR
suggests that
this receptor may play a specific role in situations of nutritional
lipid overload. In this context, it will be of interest to examine
possible connections between ligand preference, differential tissue
distribution of the PPAR subtypes, and the fatty acid content of each
tissue. It should be noted that a reverse relationship appears to exist
between affinity for ligand and overlap in ligand recognition by PPARs.
Thus, in contrast to fatty acids, the eicosanoids 8(S)-HETE
and 15d-
12,14-PGJ2, which naturally occur at
lower concentrations, are high- affinity PPAR ligands exhibiting a more
strict subtype specificity than their polyunsaturated fatty acid
precursors.
8(S)-HETE Is a High-Affinity PPAR Ligand
As mentioned above, we have identified the hydroxyeicosatetraenoic
acid 8(S)-HETE as a high-affinity ligand of the PPAR and
have confirmed previous observations that it is a strong activator of
hPPAR
(20). 8(S)-HETE is a naturally occurring compound
that is thought to result from the NADPH-dependent metabolism of
arachidonic acid by monooxygenases (cytochrome P 450) (23). Although a
considerable amount of information is available on the biological
action of the 5(S)-, 12(S)-, and
15(S)-HETE regioisomers (review in 24 , little is known
about the 8(S)-isomer. It is noteworthy, however, that
8(S)-HETE has been found in a variety of tissues
(e.g. liver, brain, epidermis) and cell types
(e.g. neutrophils, smooth muscle, oocytes) (Ref. 24 and
references therein). It is tempting to speculate that binding of
8(S)-HETE to PPAR
results in the stimulation of fatty
acid oxidative pathways that would lead to its own neutralization and
degradation, in a situation similar to that described recently for
another chemotatic eicosanoid, LTB4 (12).
Like 8(S)-HETE, the arachidonic acid analog ETYA, a compound
with very diverse biological effects (Ref. 7 and references therein),
has also been shown to be a potent xPPAR ligand. The identification
of these two high-affinity PPAR
ligands could eventually lead to the
development of medically important subtype-specific ligands and to
an increased understanding of the biological functions of PPAR
.
Bezafibrate Is a High-Affinity Ligand of xPPARß
The biological function of PPARß is the least understood of the
three PPAR subtypes despite the fact that it is the most widely
expressed one (5, 25). Here we have identified bezafibrate as the first
specific and high-affinity ligand for this receptor. Bezafibrate is
actually the only fibrate from those tested that exhibits sensus
stricto classic hormonal ligand characteristics, i.e.
subtype specificity and low ED50 both in CARLA and
transcriptional activation. It will be of interest to examine whether
PPARß from mouse and human (26, 27, 28) are also activated by this
compound as it is still an open question whether xPPARß is actually
the homolog of the mammalian PPARß (or , or NUC I) or whether it
represents a different PPAR subtype. Furthermore, the availability of a
PPARß-specific ligand provides a precious tool in investigating the
biological function of this receptor.
In contrast to bezafibrate and ciprofibrate, which have been identified
as PPAR ligands by CARLA, nafenopin, a nonligand by CARLA definition,
activates all three PPARs (Refs. 4 and 5 and present study).
Furthermore, we have observed that BRL 49653, a PPAR-specific
ligand, activates PPAR
in the HeLa cells used in this study. Thus,
it is possible that nafenopin or other fibrates and BRL 49653 activate
some PPAR subtypes indirectly, either by inducing production of
endogenous PPAR ligands or by posttranslational modification of the
receptor. Alternatively, these compounds could be modified and
converted to ligands of different PPAR subtypes in the cell. It should
also be noted that BRL 49653 has been reported not to have an effect on
mPPAR
transcriptional activity in CV-1 cells (9, 11). However, in
these studies chimeric receptors with a heterologous DNA-binding domain
were used. Thus, activation of PPAR
by these two compounds may be
cell type and/or receptor N-terminal domain-dependent. Nevertheless, as
previously mentioned, we cannot exclude the possibility that compounds
negative in CARLA are true PPAR ligands. According to this possibility,
binding of nafenopin or BRL 49653 to the receptor could cause distinct
conformational changes that do not favor PPAR/SRC-1 interactions. Thus,
the effect of these compounds in PPAR-dependent transcription could be
mediated by other coactivator(s). However, considering the ability of
fibrates (e.g. bezafibrate, ciprofibrate) to induce
favorable LBD conformation for receptor/co-activator interactions, we
think that this is an unlikely possibility. Also arguing against this
possibility is the fact that classic Scatchard analysis has failed to
identify BRL 49653 as PPAR
ligand (9).
Activation of PPAR by BRL 49653 could be consistent with the
observation that this antidiabetic compound exhibits hypolipidemic
properties as well (29). Accumulating evidence suggests that PPAR
is
the receptor subtype regulating fatty acid catabolism (reviewed in 3 , although it may also have a direct role in adipogenesis (20, 30).
Thus, it is currently believed that the activity of hypolipidemic drugs
is mediated through PPAR
. Similarly, the major function attributed
to PPAR
is that of key regulator of adipogenesis (reviewed in 31 . We have demonstrated herein that hypolipidemic agents, such as the
fibrates, are also PPARß and
ligands. Thus, the hypolipidemic
effects of these compounds may also be a result of lipid clearance via
PPAR
(storage of circulating triglycerides in adipose tissue)
and/or activation of lipid catabolism through PPARß. Future
investigation, aided by the identification of subtype-specific ligands,
will clarify whether fatty acid catabolism and adipogenesis depend on a
particular PPAR subtype or on the combined action of the three
receptors.
Conclusions
The development of the CARLA screen and the identification
of synthetic and natural PPAR ligands opens many experimental routes
for the analysis of the PPAR function. The fact that certain fatty
acids and eicosanoids can act as ligands of nuclear receptors reveals a
novel role for these molecules in the overall lipid metabolism and
cellular energy balance and implicates the function of PPARs in
processes such as transport, uptake, activation, oxidation, or storage
of fatty acids, as well as clearance of biologically very active
eicosanoids. In addition, the hypoglycemic effects of PPAR agonists
imply that these receptors may have key functions in the interface
between lipid and carbohydrate metabolism. It should be possible now,
through comparative analysis of PPAR ligand and LBD structures, to
define the structural requirements for ligand binding to this subfamily
of nuclear receptors. The subsequent development of subtype-specific
agonists and antagonists, in addition to their potential utility in
treating lipid disorders, should also provide powerful tools for
deciphering the precise role of PPARs in the metabolic pathways they
regulate.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmid Constructions
The mammalian expression vectors (pSG5) for the
Xenopus PPAR, -ß, and -
have been previously
described (5). The mPPAR
(4) was a kind gift of S. Green; the
mPPAR
1 (27) was a kind gift of J. M. Lehmann; the hPPAR
(32) was
a kind gift of F. Gonzalez; and the hPPAR
(33) was a kind gift of A.
Elbrecht and M. D. Leibowitz. All PPAR cDNAs were cloned in the pSG5
vector. The SRC-1 cDNA (17), PCR amplified from a human B cell cDNA
library, was cloned in the BamHI site of pSG5. The reporter
plasmid Cyp2XPAL contains two copies of the CYP4A6 PPRE cloned in
palindromic orientation upstream of the minimal herpes simplex virus
thymidine kinase promoter in the pBLCAT8+ plasmid (12). The GST-PPAR
LBD fusions were constructed as follows: the xPPAR
and -ß cDNA
sequences corresponding to the LBD (codons 167470 and 93397, for
and ß, respectively) were amplified by PCR and cloned into the
BamHI site of pGEX1 vector (Pharmacia, Piscataway, NJ); the
xPPAR
LBD (codons 214477) was excised as a BamHI
fragment from the ER-PPAR chimera (5) and cloned into the corresponding
site of pGEX1.
CARLA Pulldowns
One milliliter of overnight cultures of Escherichia
coli BL2 strain, transformed with the GST-PPAR LBD fusion
plasmids, was used to inoculate 50 ml of LB medium. The bacteria were
incubated at 37 C until the culture reached A600 of 0.6. At
that point, IPTG was added at final concentration of 0.1
mM, and the culture was incubated for a further 3 h at
28 C. Bacterial cells were harvested by centrifugation and suspended in
one tenth of the culture volume in NETN buffer (20 mM
Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5%
Nonidet P40) supplemented with 1 mM dithiothreitol, 0.5%
dry milk, and protease inhibitors (Complete, Boehringer Mannheim,
Indianapolis, IN). Bacteria cells were then lysed by mild sonication.
After centrifugation to remove the cell debris, glutathione Sepharose
4B beads (Pharmacia) equilibrated in NETN were added to the sonication
extracts (250 µl of beads suspension per 50 ml of culture volume),
and the GST-PPAR LBD fusions were allowed to bind to the beads for
1 h at 4 C with constant rotation. The glutathione-bound fusion
proteins were washed several times with NETN and were resuspended in
the appropriate volume of the same buffer. CARLA was performed in a
total volume of 1 ml per reaction containing 23 µg of fusion
protein. In each reaction, 20 µl of a 1:10 dilution in NETN of
35S-labeled, in vitro translated (TNT, Promega,
Madison, WI) SRC-1 were added in ice (0.2% final concentration of
translation reaction mix). The compounds tested were added at the
appropriate concentrations. Probucol and nervonic acid initially
precipitated in ice but were completely dissolved at 4 C. After
incubation of the reaction mixture for 4 h at 4 C with constant
rotation, the immobilized fusion proteins were washed three times with
0.5 ml NETN, without milk. The glutathione-Sepharose-bound proteins
were dried under vacuum before being resuspended in SDS sample buffer
and subjected to SDS-PAGE. Before being dried and exposed to
autoradiography, the polyacrylamide gels were lightly stained with
Coomasie brilliant blue to control that equal quantities of fusion
proteins were used in each reaction. The amounts of retained SRC-1 were
determined by image analysis on a GS 250 molecular Imager (Bio-Rad
Laboratories, Richmond, CA) instrument.
Cell Culture and Transfections
Cell culture conditions and transfections of HeLa cells have
been previously described (7). For each transfected plate (3 cm
diameter), 2.5 x 105 cells and 0.1 µg PPAR
expression plasmid were used along with 2 µg of the reporter and 0.5
µg of the CMV-ß-gal (34) internal control plasmid. Twelve hours
posttransfection, cells were incubated for a further 24 h with
culture medium containing the ligands.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
This work was supported by the Swiss National Science Foundation and the Etat de Vaud. G. K. was supported by Ciba-Geigy. E. K. is a Netherlands Organization for Scientific Research (N.W.O.) fellow.
Received for publication January 30, 1997. Accepted for publication March 17, 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|