Abstract:
Peroxisome proliferator-activated receptors (PPARs) are members of a
large group of ligand-regulated transcription factors that includes nuclear
receptors for steroid and thyroid hormones, retinoids and vitamin D₃.
Synthetic fibrates and thiazolidinediones that bind to and activate PPARs are
used efficaciously in humans to remedy hypertriglyceridemia and non-insulin
dependent diabetes mellitus, respectively. The objective of the studies
described herein was to elucidate the molecular mechanisms of ligand-dependent
PPAR signaling.
Several PPAR ligands, including WY-14,643, were demonstrated to
directly induce PPARα conformational changes as evidenced by a differential
protease sensitivity assay. Conformational changes were induced in a dose-dependent
manner which paralleled that of ligand to induce transcriptional
activation. Direct interaction of ligands with, and the resulting conformational
alterations in, PPARα may facilitate interaction of the receptor with
transcriptional intermediary factors and thus may underlie the molecular basis
of ligand-dependent transcriptional activation mediated by PPARα.
The yeast two hybrid screen was utilized to identify downstream
components of the PPARα signaling pathway. Using this technique, the
coactivator proteins, p300 and steroid receptor coactivator-1 (SRC-1), were
identified as PPARα-interacting proteins and WY-14,643 potentiated these
interactions. p300 also enhanced the transcriptional activation properties of
PPARα and, therefore, can be considered a bona fide coactivator for this
nuclear receptor. Nuclear receptor corepressor (NCoR) was also isolated as
a PPARα-interacting protein from a yeast two hybrid screen. In contrast to the
ligand enhanced PPARα-coactivator interactions, WY-14,643 inhibited NCoR
interaction with PPARα. NCoR and the coactivators, p300 and SRC-1, were
also demonstrated to require distinct receptor regions for efficient interaction
with PPARα.
Results described herein demonstrate that ligand induces PPARα
conformational changes, promotes PPARα-coactivator (p300 and SRC-1)
interactions, and inhibits PPARα-NCoR interactions. We hypothesize that
such molecular events are critical for ligand-dependent transcriptional
activation by PPARα. These results contribute additional knowledge as to the
molecular mechanisms of PPAR-dependent signaling and may act as a
starting part for the improvement and/or development of therapeutic strategies
aimed at manipulating this signaling pathway.
