Major intestinal coactivator p300 strongly activates peroxisome proliferator-activated receptor in intestinal cell line, Caco-2

Abstract

We have previously reported that several genes related to intestinal fatty acid and vitamin A metabolism are coordinately regulated by peroxisome proliferator-activated receptor (PPAR) [Arch. Biochem. Biophys. 389 (2001) 41; Biochim. Biophys. Acta 1531 (2001) 68]. In this study, we demonstrated that PPARα and PPARδ interacted with endogenous coactivators in intestinal cell line, Caco-2 in a ligand specific manner. We isolated rat cDNA clones encoding the nuclear receptor interaction domains of the two transcriptional coactivators, CREB-binding protein (CBP) and p300. Expression level of CBP mRNA was relatively low in the small intestine, while p300 mRNA was ubiquitously expressed in various tissues including the small intestine in the rat. Southern blot analysis revealed that these coactivators were encoded by different genes. Mammalian two-hybrid assays in Caco-2 cells revealed that p300 interacted with PPARα or PPARδ in the presence of their specific ligands more efficiently than CBP did. These results suggest that the major intestinal coactivator, p300 strongly interacts with PPARα and PPARδ.

Introduction

Nuclear receptor superfamily exert its function by binding to specific DNA response elements within the regulatory regions of target genes and by modulating expression of these genes at the transcriptional level (O'Malley and Tsai, 1992, Beato et al., 1995, Mangelsdorf et al., 1995). The nuclear receptors are composed of three domains: a N-terminal region containing a transactivation function (AF-1), a DNA binding-domain (DBD) harboring two zinc finger motifs, and the C-terminal ligand binding-domain (LBD), which contains the dimerization interface and a second activation domain (AF-2) (Mangelsdorf et al., 1995). AF-1 is capable of activating transcription in a ligand-independent manner (Mangelsdorf et al., 1995, Gelman et al., 1999). The AF-2 domain overlaps the C-terminal ligand-binding domain (LBD) and activates transcription in response to ligands (Mangelsdorf et al., 1995). While the mechanisms by which the AF-2 domain transmits ligand binding signals to basal transcriptional machinery remain poorly understood, many groups have identified several types of transcriptional coactivators that interact with the AF-2 domain of various nuclear receptors in a ligand-dependent manner (Glass and Rosenfeld, 2000, McKenna et al., 1999, Freedmann, 1999). It appears that nuclear receptor AF-2 domain exerts a function via the establishment of a multiprotein co-activator complex comprising the receptor, member(s) of the SRC-1/TIF2 coactivator family, CBP/p300 coactivator family, and the CBP-binding factor p/CAF (Moras and Gronemeyer, 1998). These coactivator complexes have been shown to possess histone acetyltransferase activity, and it is believed that chromatin disruption is a critical, albeit apparently insufficient, step in the nuclear receptor-dependent gene activation (Goodman and Smolik, 2000).

Peroxisome proliferator-activated receptors (PPARs) are the lipid-activated transcription factors that belong to the nuclear hormone receptor superfamily (Desvergne and Wahli, 1999). To date, three subtypes of PPARs have been cloned in amphibians, rodents, and humans: PPARα, PPARδ (also called β, NUC-1, or FAAR), and PPARγ (Issemann and Green, 1990, Gottlicher et al., 1992, Xing et al., 1995, Elbrecht et al., 1996). Various types of fatty acids, some eicosanoids, and some hypolipidemic and antidiabetic drugs have been shown to activate PPARs as their ligands (Forman et al., 1995, Forman et al., 1997, Kliewer et al., 1997).

Our previous study showed that several genes related to β-oxidation of fatty acids and those related to intracellular transfer of fatty acids and vitamin A are regulated by PPAR in the small intestine (Mochizuki et al., 2001a, Mochizuki et al., 2001b, Suruga et al., 1999a, Suruga et al., 1999b). However, it is unclear between CBP and p300 which plays a major role in coactivation of PPAR-dependent genes in the small intestine. Therefore, we first cloned the nuclear receptor-interaction domain of rat CBP and rat p300, and then we examined whether CBP and p300 differentially interacted with the two major subtypes of PPARs expressed in the small intestine, i.e. PPARα and PPARδ.