(Received for publication, August 13, 1996, and in revised form, November 15, 1996)
From the Departments of Molecular Endocrinology and
§ Cell Biology, Glaxo Wellcome Research and Development,
Research Triangle Park, North Carolina 27709 and ¶ Affymax,
Santa Clara, California 95051
Indomethacin is a non-steroidal anti-inflammatory
drug (NSAID) and cyclooxygenase inhibitor that is frequently used as a
research tool to study the process of adipocyte differentiation.
Treatment of various preadipocyte cell lines with micromolar
concentrations of indomethacin in the presence of insulin promotes
their terminal differentiation. However, the molecular basis for the
adipogenic actions of indomethacin had remained unclear. In this
report, we show that indomethacin binds and activates peroxisome
proliferator-activated receptor (PPAR
), a ligand-activated
transcription factor known to play a pivotal role in
adipogenesis. The concentration of indomethacin required to activate
PPAR
is in good agreement with that required to induce the
differentiation of C3H10T1/2 cells to adipocytes. We demonstrate that
several other NSAIDs, including fenoprofen, ibuprofen, and flufenamic
acid, are also PPAR
ligands and induce adipocyte differentiation of
C3H10T1/2 cells. Finally, we show that the same NSAIDs that activate
PPAR
are also efficacious activators of PPAR
, a liver-enriched
PPAR subtype that plays a key role in peroxisome proliferation.
Interestingly, several NSAIDs have been reported to induce peroxisomal
activity in hepatocytes both in vitro and in
vivo. Our findings define a novel group of PPAR
ligands and
provide a molecular basis for the biological effects of these drugs on
adipogenesis and peroxisome activity.
Indomethacin and other NSAIDs1 are used clinically for their anti-inflammatory, anti-pyretic, and analgesic properties (1). The molecular basis for the therapeutic actions of NSAIDs is believed to be their ability to inhibit cyclooxygenase (COX) activity and thereby block the production of prostaglandins (PGs). Two COX enzymes have been identified. COX-1 is constitutively expressed, and the PGs produced by this enzyme are thought to function in the so-called housekeeping functions of the cell; in contrast, the COX-2 isozyme is an inducible enzyme that is normally absent from cells but is expressed in response to growth factors, tumor promoters, and cytokines (2). Most of the NSAIDs inhibit both COX-1 and COX-2, although they vary in their relative potencies against the two COX isozymes (3).
Indomethacin is also widely used as a research tool to study the process of adipocyte differentiation. While there is at least one report of indomethacin blocking adipocyte differentiation (4), treatment of several preadipocyte cell lines with this drug results in their terminal differentiation (5-7). Early reports suggested that indomethacin might function as an adipogenic agent through its inhibition of COX activity. However, two lines of evidence indicate that the adipogenic activity of indomethacin cannot simply be ascribed to the inhibition of COX. First, the concentration of drug required to induce differentiation is 2-3 orders of magnitude higher than that required to inhibit COX activity, and second, several NSAIDs that inhibit COX activity fail to induce adipocyte differentiation (7). Thus, the mechanism underlying the adipogenic activity of indomethacin has remained obscure.
Insight into the molecular mechanisms responsible for adipocyte
differentiation was recently provided by the identification of a
ligand-activated transcription factor, termed PPAR, as a key
regulator of adipogenesis (8). PPAR
, a member of the nuclear receptor superfamily, is selectively expressed in adipocytes and induced early during the course of differentiation of several preadipocyte cell lines (9, 10). Forced expression of PPAR
in
fibroblast and myoblast cell lines results in efficient adipocyte differentiation in a PPAR
-activator-dependent fashion
(8, 11). Thus, PPAR
functions as a master regulator of adipocyte differentiation. Two other PPAR subtypes, termed PPAR
and PPAR
, have been identified in addition to PPAR
(12, 13). PPAR
is the
predominant PPAR subtype expressed in liver and is activated by a group
of chemicals that induce the proliferation of peroxisomes in rodents
(14). Gene disruption experiments have demonstrated that PPAR
is
required for the pleiotropic hepatic response to peroxisome
proliferators in rodents (15).
Work from several laboratories had shown that PGs have marked effects,
both positive and negative, on adipocyte differentiation (4, 16-19).
Interestingly, PPAR is activated by PGs and PG-like molecules
(20-23). Recently, the arachidonic acid metabolite
15-deoxy-
12,14-PGJ2 was shown to bind
directly to PPAR
and to promote the efficient conversion of
fibroblast and mesenchymal stem cell lines to adipocytes (24, 25). The
finding that a PG functions as a PPAR
ligand and promotes adipocyte
differentiation provided additional evidence that products of the COX
pathway play an important role in modulating adipogenesis.
If prostanoids can function as PPAR ligands and induce adipogenesis,
how then does a COX inhibitor such as indomethacin, which blocks PG
synthesis, promote adipocyte differentiation? In this report, we show
that indomethacin and several other NSAIDs function as PPAR
ligands,
suggesting a mechanism for the adipogenic actions of these compounds.
Furthermore, we demonstrate that these same NSAIDs also activate
PPAR
, providing a basis for the reported effects of NSAIDs on
peroxisome activity in liver.
Indomethacin, flufenamic acid, fenoprofen,
ibuprofen, piroxicam, acetaminophen, and salicylic acid were purchased
from Sigma. The peroxisome proliferator Wy14,643 was
purchased from Biomol (Plymouth Meeting, PA) and
15-deoxy-12,14-PGJ2 from Cayman Chemical
Company (Ann Arbor, MI).
To generate the pSG5-GAL4-PPARLBD
and pSG5-GAL4-PPAR
LBD chimeric receptor expression plasmids,
cDNAs encoding the ligand binding domains (LBDs) of the human
PPAR
(amino acids 167-468) (26) and the human PPAR
(amino acids
176-477) (27) were amplified by polymerase chain reaction and
subcloned into the pSG5-GAL4 expression plasmid (28). The pCMV-PPAR
expression plasmid has been described (27). The
(UAS)5-tk-CAT and aP2-tk-CAT reporter plasmids were
previously described (28). Transient cotransfection assays using these
plasmids were performed as described previously (28).
The LBD of human PPAR (amino acids
176-477) (27) was overexpressed in Escherichia coli as a
histidine-tagged fusion protein and bacterial lysates prepared as
described previously (25). For competition binding assays, bacterial
extracts (approximately 100 µg of protein) containing the PPAR
ligand binding domain were incubated at 4 °C for 2-3 h with 40 nM [3H]BRL49653 (specific activity, 40 Ci/mmol) in the absence or presence of unlabeled competitor in buffer
containing 10 mM Tris (pH 8.0), 50 mM KCl, 10 mM dithiothreitol. Bound was separated from free radioactivity by elution through 1- ml Sephadex G-25 desalting columns
(Boehringer Mannheim). Bound radioactivity eluted in the column void
volume and was quantitated by liquid scintillation counting. Data shown
are the result of binding assays performed in duplicate, and each
experiment was repeated at least twice with similar results.
C3H10T1/2 clone 8 murine fibroblasts (American Type
Culture Collection) were maintained in Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) supplemented with 10% fetal calf
serum and 10 µg/ml penicillin and streptomycin. One day after
reaching confluence, the cells were treated with BRL49653 or the
various NSAIDs in the presence of 200 nM insulin. Fresh
media and test compounds were added every 2 days. Lipogenesis was
measured in cells at 9 days post-confluence as described previously
(29). For Northern analysis, total RNA was prepared from vehicle- and compound-treated cells using the RNeasy Total RNA Kit (Qiagen, Chatsworth, CA). Fifteen µg of total RNA was electrophoresed on a
formaldehyde gel and transferred to nitrocellulose. The blot was probed
with mouse aP2 and GAPDH probes labeled via the random priming
technique with [-32P]dCTP. The results of Northern
assays were quantitated using a Molecular Dynamics Computing
Densitometer and Image Quant software.
The PPARs are activated by a
large number of structurally diverse compounds including prostanoids,
long-chain fatty acids, the fibrate class of hypolipidemic drugs,
leukotriene antagonists, and anti-diabetic thiazolidinediones (13).
While chemically diverse, these compounds share certain structural
characteristics including a lipophilic backbone and an acid moiety,
usually a carboxylate. Indomethacin and many of the other NSAIDs are
amphipathic carboxylates that share these broad structural features
(Fig. 1). This suggested to us that indomethacin might
exert its adipogenic effects through direct activation of PPAR.
We tested the possibility that indomethacin activates PPAR via a
transient transfection assay. An established chimera system was used
(28) in which the LBD of PPAR
was fused to the DNA binding domain of
the yeast transcription factor GAL4. The advantage of the GAL4 chimera
assay is that it minimizes background due to the cell's endogenous
receptors. Expression plasmid for the GAL4-PPAR
LBD chimera was
transfected into CV-1 cells together with a reporter construct
containing five copies of the GAL4 response element driving expression
of the reporter chloramphenicol acetyltransferase (UAS5-tk-CAT). Dose-response analysis revealed that
indomethacin is an efficacious activator of PPAR
, inducing PPAR
activity roughly 40-fold at 1 × 10
4 M
(Fig. 2). This activation is comparable with the maximal
induction obtained with the PPAR
ligands
15-deoxy-
12,14-PGJ2 and the anti-diabetic
thiazolidinedione BRL49653 (25, 28) (see below). Indomethacin activated
PPAR
with an EC50 of approximately 4 × 10
5 M (Fig. 2). Similar EC50 and
fold activation values were obtained for indomethacin in transient
transfection assays performed with an expression vector for wild-type
PPAR
and a reporter driven by the fatty acid binding protein/aP2
enhancer region which contains two PPAR
response elements (10) (Fig.
2). Thus, indomethacin is an efficacious activator of PPAR
.
Indomethacin Binds PPAR
We next sought to determine
whether indomethacin activates PPAR through direct interactions with
the receptor. We and others (24, 28) have previously shown that the
anti-diabetic thiazolidinedione BRL49653 can bind to PPAR
with high
affinity. The ability of indomethacin to bind to PPAR
was assessed
in a competition binding assay using [3H]BRL49653 and
bacterially expressed PPAR
LBD. As shown in Fig. 3,
indomethacin competed efficiently with [3H]BRL49653 for
binding to the PPAR
LBD, with an IC50 of approximately 1 × 10
4 M. In control experiments,
acetaminophen, an NSAID that does not activate PPAR
(see below),
failed to compete with [3H]BRL49653 for binding to the
PPAR
LBD (Fig. 3). These data demonstrate that indomethacin can
interact directly and specifically with the PPAR
LBD and thus define
a novel PPAR
ligand.
Other Classes of NSAIDs Activate PPAR
Several
chemically distinct classes of NSAIDs are used clinically including
thiazinecarboxamides (e.g. piroxicam) and derivatives of
arylacetic acid (e.g. indomethacin), aminoarylcarboxylic
acid (e.g. flufenamic acid), arylpropionic acid
(e.g. ibuprofen and fenoprofen), and salicylic acid
(e.g. aspirin) (Fig. 1) (1). We next tested whether
representative compounds from the different classes of NSAIDs could
also activate PPAR. CV-1 cells were transfected with the
GAL4-PPAR
LBD expression plasmid and the UAS5-tk-CAT reporter and treated with 1 × 10
4 M of
piroxicam, flufenamic acid, ibuprofen, fenoprofen, and salicylic acid.
As shown in Fig. 4A, flufenamic acid,
fenoprofen, and ibuprofen were efficient activators of PPAR
,
activating the receptor to a degree comparable to that obtained with
the PPAR
ligands BRL49653 and
15-deoxy-
12,14-PGJ2 and the peroxisome
proliferator Wy14,643. However, in contrast to indomethacin, no
activation of PPAR
was observed in transfected cells treated with
1 × 10
5 M of these compounds (data not
shown). Thus, indomethacin is the most potent of the NSAIDs that we
tested for PPAR
activation. Treatment of transfected cells with
piroxicam resulted in only a modest activation of PPAR
(approximately 5-fold), whereas treatment with salicylic acid or
acetaminophen resulted in little or no induction of reporter expression
(Fig. 4A). We note that the compounds that activated PPAR
efficiently (>6-fold) were all amphipathic acids (Fig. 1) and thus
conform in their general structural features to known PPAR
activators.
The ability of these compounds to interact with PPAR was assessed in
the competition binding assay using [3H]BRL49653. These
studies revealed a good correlation between the compounds that
activated PPAR
in the transfection assay and those that interacted
directly with the receptor. Although flufenamic acid, fenoprofen, and
ibuprofen competed efficiently with [3H]BRL49653 for
binding to the PPAR
LBD, little or no competition was seen with
piroxicam, salicylic acid, or acetaminophen (Fig. 4B). Taken
together, the transfection and binding analyses demonstrate that some
but not all NSAIDs bind and activate PPAR
.
For comparative purposes, we also tested the various NSAIDs on the
PPAR and PPAR
subtypes using the transfection assay. Little or no
activation of PPAR
was seen in the presence of 1 × 10
4 M of these compounds (data not shown).
However, indomethacin, fenoprofen, ibuprofen, and flufenamic acid were
efficacious activators of PPAR
at this concentration, with
fenoprofen activating the receptor to a degree comparable to that
obtained with the strong peroxisome proliferator Wy14,643 (Fig.
4A). Thus, the same NSAIDs that activate PPAR
are also
efficacious activators of the PPAR
subtype.
We and others (24,
25, 28, 30-33) have shown that treatment of various fibroblast and
mesenchymal stem cell lines with PPAR ligands, including
15-deoxy-
12,14-PGJ2 and the anti-diabetic
thiazolidinediones, promotes their efficient conversion to adipocytes.
As discussed, indomethacin is used to promote the terminal
differentiation of preadipocyte cell lines. We next examined whether
the concentration of indomethacin required to activate PPAR
in CV-1
cells was consistent with that required to induce adipocyte
differentiation. C3H10T1/2 mouse mesenchymal stem cells were treated
with various concentrations of indomethacin and subsequently assayed
for lipogenesis, an established measure of adipocyte differentiation
(29). Dose-response analysis revealed the EC50 for
indomethacin in the lipogenesis assay to be approximately 8 × 10
5 M (Fig. 5A).
This value is in good agreement with that reported in a previous study
(3 × 10
5 M) using TA1 cells, a stable
adipogenic cell line derived from C3H10T1/2 cells (7), and is also
consistent with the EC50 value of indomethacin for PPAR
activation in the transfection assay (Fig. 2). Taken together, these
data suggest that PPAR
is the target for the adipogenic actions of
indomethacin.
Our finding that flufenamic acid, fenoprofen, and ibuprofen also
activated PPAR at micromolar concentrations suggested that these
NSAIDs might also promote adipocyte differentiation. We tested this
possibility using C3H10T1/2 cells and the lipogenesis assay. In
agreement with previous studies, treatment of the C3H10T1/2 cells with
either 1 × 10
6 M of the
thiazolidinedione BRL49653 or 3 × 10
6 M
15-deoxy-
12,14-PGJ2 resulted in marked
increases in adipocyte differentiation (Fig. 5B) (25, 28).
As expected, concentrations of the hypolipidemic agent Wy14,643
sufficient to activate PPAR
also induced lipogenesis (Fig.
5B) (9, 28). Treatment of C3H10T1/2 cells with 1 × 10
4 M of either flufenamic acid or fenoprofen
promoted lipogenesis, albeit less efficiently than indomethacin (Fig.
5B). The results of the lipogenesis assay were confirmed by
oil red O staining for lipid accumulation in treated cells (data not
shown). The NSAIDs that did not activate PPAR
efficiently in the
transfection assay, including piroxicam, salicylic acid, and
acetaminophen, failed to induce lipogenesis in the C3H10T1/2 cells
(Fig. 5B). We conclude that NSAIDs other than indomethacin
can also promote adipocyte differentiation at concentrations at which
they activate PPAR
.
We note that ibuprofen, which was a less efficacious activator of
PPAR in CV-1 cells than either flufenamic acid or fenoprofen (Fig.
4A), failed to promote lipogenesis in C3H10T1/2 cells when tested at 1 × 10
4 M (Fig.
5B). However, increasing the concentration of indomethacin to 5 × 10
4 M resulted in significant
lipogenesis (Fig. 5B). In Northern analysis, 1 × 10
4 M ibuprofen induced weak expression of
the gene encoding aP2, an adipocyte-specific fatty acid binding protein
whose expression is directly regulated by PPAR
(Fig. 5C)
(10). Consistent with the results of the transfection studies, 1 × 10
4 M indomethacin stimulated aP2 gene
expression approximately 3-fold more efficiently than ibuprofen (Fig.
5C). Taken together, these data indicate that ibuprofen is
less potent than indomethacin, flufenamic acid, or fenoprofen in the
activation of PPAR
in both CV-1 and C3H10T1/2 cells.
The NSAID indomethacin is frequently included as one of a mixture
of compounds used to promote the terminal differentiation of various
preadipocyte cell lines in vitro. This differentiation mixture also routinely includes insulin, corticosteroids, and isobutylmethylxanthine. Recent work has indicated that
isobutylmethylxanthine and corticosteroids induce the expression of the
genes encoding CCAAT/enhancer binding proteins and
,
respectively, members of the basic region-leucine zipper family of
transcription factors (34). These two transcription factors are induced
early during the course of 3T3-L1 cell conversion to adipocytes and
appear to play key roles in the differentiation cascade (34-37). The
mechanism underlying the adipogenic activity of indomethacin, however,
has remained unclear. Early speculation focused on the ability of indomethacin to inhibit COX activity. However, Knight et al.
(7) showed that the concentration of indomethacin required to promote the differentiation of TA1 cells to adipocytes was 1-2 orders of
magnitude greater than the concentrations needed to block prostaglandin synthesis. Furthermore, not all COX inhibitors promoted adipocyte differentiation. Likewise, we have found that several COX inhibitors, including the potent NSAID piroxicam (38), fail to promote adipocyte differentiation. These data provide compelling evidence that the effects of indomethacin are not mediated through the inhibition of
prostaglandin production.
In searching for the basis of its adipogenic activity, we have found
that indomethacin functions as a micromolar ligand for the adipogenic
transcription factor PPAR. PPAR
is abundantly expressed in
adipose tissue where it functions as a key modulator of the adipocyte
differentiation program (8-10). PPAR
ligands, including the
anti-diabetic thiazolidinediones and the arachidonic acid metabolite
15-deoxy-
12,14-PGJ2, are potent inducers of
the differentiation of several different fibroblastic cell lines to
adipocytes (30-33). Our finding that the concentration of indomethacin
required to induce C3H10T1/2 cell differentiation correlates with that
required to activate PPAR
in the transfection assay provides strong
evidence that the adipogenic actions of this NSAID are mediated through
its binding and activation of PPAR
.
While indomethacin is widely used to promote the differentiation of
preadipocyte cell lines, in at least one instance indomethacin was
found to block this process (4). Our results together with the recent
findings that a subset of the PGs activate PPAR (23-25) may provide
an explanation for this paradox. At lower concentrations, indomethacin
may block COX activity, thus inhibiting the formation of adipogenic PGs
and activators of PPAR
such as
15-deoxy-
12,14-PGJ2 and prostacyclin without
directly affecting the PPARs. At higher concentrations, however,
indomethacin not only inhibits COX activity but also acts as a PPAR
agonist, promoting adipocyte differentiation. Thus, indomethacin may
function to either inhibit or induce adipogenesis depending upon the
particular concentration of drug used in the experiment.
We have shown that the same NSAIDs that activate PPAR also activate
PPAR
. PPAR
is the predominant PPAR subtype expressed in the
rodent liver (14, 21). Targeted gene disruption experiments have shown
that PPAR
is essential for the induction of peroxisomal enzymes and
peroxisome proliferation in the rodent liver (15). Interestingly,
several NSAIDs have been reported to have marked effects on peroxisome
activity in hepatocytes when used either in vitro or
in vivo. Indomethacin and ibuprofen induced
-oxidation in
peroxisomes of cultured hepatocytes (39). Furthermore, treatment of
rats with ibuprofen induced peroxisomal
-oxidation, reduced serum
triglycerides and cholesterol, and increased liver weight (39).
Finally, treatment of rats with benoxaprofen, an NSAID closely related
to ibuprofen, at doses comparable to those used clinically induced
peroxisomal
-oxidation and increased cytochrome P4504A1 apoprotein
and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional
protein levels in liver (40). Expression of both of these genes is
known to be induced by PPAR
in response to peroxisome proliferators
(15, 41, 42). As a whole, the effects of these NSAIDs on liver function
are similar to those seen in experiments performed with the fibrate
class of hypolipidemic drugs (43), agents that are established
activators of PPAR
(14). Our data strongly suggest that the actions
of NSAIDs on peroxisomal and liver functions are mediated through the
activation of PPAR
. We note, however, that we do not have a binding
assay for mammalian PPAR
and, as a consequence, have not been able to demonstrate direct interactions between the NSAIDs and PPAR
. Thus, it remains possible that the NSAIDs modulate PPAR
activity through an indirect mechanism. It is interesting that NSAIDs are associated with a variety of detrimental side effects, including hepatotoxicity (1). While there is currently no evidence of a link
between these negative effects and PPARs, evaluation of the activities
of NSAIDs on PPARs may be useful in minimizing the potential for
unwanted side effects as new drugs of this class are developed.
In summary, we have demonstrated that indomethacin and other NSAIDs are
efficacious activators of PPAR and PPAR
at micromolar concentrations. These data provide evidence for a common mechanism underlying the seemingly disparate biological effects of these compounds on the induction of adipocyte differentiation in
vitro and peroxisome proliferation in vivo.
We thank Tim Willson for preparation of Fig. 1 and critical reading of the manuscript and Larry Hammacher for assistance with cell culture.