Ligand-independent Activation Domain in the N Terminus of
Peroxisome Proliferator-activated Receptor (PPAR)
DIFFERENTIAL ACTIVITY OF PPAR1 AND -2 ISOFORMS AND INFLUENCE OF INSULIN*
(Received for publication, April 8, 1997, and in revised form, May 19, 1997)
Ariel Werman, Anthony Hollenberg, Gemma Solanes, Christian Bjo rbæk,
Antonio J. Vidal-Puig‡, and Jeffrey S. Flier§
From the Department of Medicine, Division of Endocrinology, Beth Israel Deaconess Medical Center,
Boston, Massachusetts 02215
Peroxisome proliferator-activated receptor (PPAR)
is a member of the nuclear hormone receptor superfam-
ily, and is an important regulator of adipogenesis and
adipocyte gene expression. PPAR exists as two isoforms,
PPAR1 and PPAR2, that differ only in their N termini.
Both isoforms are activated by ligands that include the
antidiabetic thiazoladinedione drugs and 15-deoxy-
12, 14
-
prostaglandin J2, and potential differences in their func-
tion have yet to be described. We report that, in addition
to a ligand-activated transcriptional activity, when stud-
ied under conditions of ligand depletion, intact PPAR
has a ligand-independent activation domain. To identify
the basis for this ligand-independent activation, we used
GAL4-PPAR chimeric expression constructs and UAS-
TK-LUC in CV1 cells and isolated rat adipocytes. In both
cell systems, isolated PPAR1 and PPAR2 N termini
have activation domains, and the activation function of
PPAR2 is 5– 6-fold greater than that of PPAR1. Insulin
enhances the transcriptional effect mediated by both
PPAR1 and PPAR2 N-terminal domains. These data
demonstrate that 1) PPAR has an N-terminal (ligand-
independent) activation domain; 2) PPAR1 and PPAR2
N termini have distinct activation capacities; and 3) insu-
lin can potentiate the activity of the N-terminal domain of
PPAR.
The peroxisome proliferator-activated receptor (PPAR)
1
is
a member of the nuclear receptor superfamily that plays a
pivotal role in the molecular determination of adipogenesis and
the regulation of adipocyte gene expression (1–5). Under ap-
propriate conditions, expression of PPAR through retroviral
infection of fibroblastic cell lines is sufficient to cause differen-
tiation along an adipocyte lineage, as assessed by expression of
adipocyte-specific genes, accumulation of lipid, and acquisition
of adipocyte morphology (6). Recently, it has been shown that
15-deoxy-
12, 14
-prostaglandin J2 (PG J2) is a high affinity
ligand for PPAR (7, 8) and that PPAR is also the receptor for
the thiazoladinedione class of insulin-sensitizing drugs (7, 8).
PPAR resembles other members of the nuclear receptor su-
perfamily in that ligand-dependent receptor activation alters
the rates of transcription of genes, specifically those that have
peroxisome proliferator response elements (PPREs) within
their promoters (e.g. aP2, phosphoenolpyruvate carboxykinase,
and uncoupling protein) (9 –11).
PPAR exists as two isoforms, PPAR1 and PPAR2, that
differ only in their N termini, with PPAR2 having an addi-
tional 30 amino acids that are encoded by a single exon (9, 12).
Expression of mRNA encoding the two isoforms is driven by
alternative promoters within a single PPAR gene (12), and
their expression is differentially regulated in a tissue-specific
manner. PPAR2 is most abundantly expressed in adipocytes
and is relatively specific for this tissue (9, 13). In contrast,
while PPAR1 is also expressed at a high level in adipocytes, it
is also found at significant but lower levels in a number of other
tissues, including muscle (13–15). Considering the relative
abundance of PPAR1 in many nonadipose tissues, it is likely
that this isoform is capable of subserving roles apart from
regulation of adipogenesis. In addition, although no functional
differences between the 1 and 2 isoforms have been described
to date, it is possible that these isoforms subserve different
functions under some conditions. However, deletion of the N-
terminal 129 amino acids of PPAR did not diminish the adi-
pogenic potency of PPAR that was introduced into 3T3 fibro-
blasts by retroviral infection (6), and it has therefore been
viewed as unlikely that the N terminus of PPAR subserves a
functionally important role.
Here, we provide evidence that PPAR, in addition to being
activated in a ligand-dependent manner, can also be activated
in a ligand-independent manner, and we define a ligand-inde-
pendent activation domain within the N terminus of PPAR.
We also demonstrate the first potential functional difference
between the two PPAR isoforms, wherein the N terminus of
PPAR2 more potently activates a heterologous promoter than
does PPAR1. We have mapped the overall activation domain
to a region common to the two isoforms and demonstrate that
the 30 amino acids unique to PPAR2 can activate a heterolo-
gous promoter only in concert with the main N-terminal acti-
vation domain. Finally, we provide evidence that the ligand-
independent activation function of the N terminus of PPAR is
augmented when cells are treated with insulin. We propose a
model for the activation of PPAR and discuss its possible
implications.
MATERIALS AND METHODS
Plasmid Construction—The PPRE reporter construct consisted of
two copies of the DR1 element upstream of the TK109 promoter in the
vector pA
3
Luc (16). All GAL4 constructs were constructed by inserting
* This work was supported by National Institutes of Health Grant
DK 28082 (to J. S. F.) and Grants DK 02354 and 46200 (to A. H.). The
costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked “adver-
tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
‡ Supported by the Paul Dudley White Fellowship Award from the
American Heart Association, Massachusetts Affiliate.
§ To whom correspondence should be addressed: Dept. of Medicine,
Division of Endocrinology, Beth Israel Deaconess Medical Center, Re-
search North 325, 99 Brookline Ave., Boston, MA 02215. Tel.: 617-667-
2151; Fax: 617-667-2927; E-mail: jflier@bidmc.harvard.edu.
1
The abbreviations used are: PPAR, peroxisome proliferator-acti-
vated receptor; PPRE, peroxisome proliferator response element; PCR,
polymerase chain reaction; TK, thymidine kinase; DBD, DNA binding
domain.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 32, Issue of August 8, pp. 20230 –20235, 1997
© 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
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