The Human Peroxisome Proliferator-activated Receptor δ Gene is a Primary Target of 1α,25-Dihydroxyvitamin D3 and its Nuclear Receptor

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Peroxisome proliferator-activated receptor (PPAR) δ is the most widely expressed member of the PPAR family of nuclear receptor fatty acid sensors. Real-time PCR analysis of breast and prostate cancer cell lines demonstrated that PPARδ expression was increased 1.5 to 3.2-fold after three hours stimulation with the natural vitamin D receptor (VDR) agonist, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In silico analysis of the 20 kb of the human PPARδ promoter revealed a DR3-type 1α,25(OH)2D3 response element approximately 350 bp upstream of the transcription start site, which was able to bind VDR-retinoid X receptor (RXR) heterodimers and mediate a 1α,25(OH)2D3-dependent upregulation of reporter gene activity. Chromatin immuno-precipitation assays demonstrated that a number of proteins representative for 1α,25(OH)2D3-mediated gene activation, such as VDR, RXR and RNA polymerase II, displayed a 1α,25(OH)2D3-dependent association with a region of the proximal PPARδ promoter that contained the putative DR3-type VDRE. This was also true for other proteins that are involved in or are the subject of chromatin modification, such as the histone acetyltransferase CBP and histone 4, which displayed ligand-dependent association and acetylation, respectively. Finally, real-time PCR analysis demonstrated that 1α,25(OH)2D3 and the synthetic PPARδ ligand L783483 show a cell and time-dependent interference in each other's effects on VDR mRNA expression, so that their combined application shows complex effects on the induction of VDR target genes, such as CYP24. Taken together, we conclude that PPARδ is a primary 1α,25(OH)2D3-responding gene and that VDR and PPARδ signaling pathways are interconnected at the level of cross-regulation of their respective transcription factor mRNA levels.

Introduction

Nuclear receptors comprise the largest family of metazoan transcription factors (of which there are 48 human members) and regulate the expression of target genes that affect diverse processes, such as reproduction, development and metabolism, by transducing the effects of small, lipophilic compounds into transcriptional responses.1 Certain nuclear receptor superfamily members have been shown to play important roles in diseases, such as type 2 diabetes, atherosclerosis, osteoporosis and cancer.2 Examples are the vitamin D3 receptor (VDR), which is the only nuclear protein that binds 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) with high affinity (Kd=0.1 nM),3 and the three peroxisome proliferator-activated receptors (PPARs) α, δ and γ, which are activated by physiological concentrations (Kd=1–100 μM) of native and oxidized polyunsaturated fatty acids as well as arachidonic acid derivatives, such as prostaglandins and prostacyclins.4 PPARδ is the only PPAR subfamily member that is widely expressed and in many tissues it is often co-expressed with VDR.5 PPARδ is overexpressed in tumors of the colon and other tissues and it was initially assumed that the receptor promotes colon polyp formation.6, 7 However, this original finding has been challenged by new evidence that shows that PPARδ may attenuate colon and skin carcinogenesis.8, 9 The latter observations appear similar to the described anti-proliferative effects of 1α,25(OH)2D3 and its synthetic analogues in a number of different types of cancer, including those of the colon, breast and prostate.10

An essential prerequisite for the direct modulation of a gene's transcription by 1α,25(OH)2D3 is the location of at least one activated VDR molecule close to the basal transcriptional machinery of a primary 1α,25(OH)2D3-responding gene. This is traditionally achieved through the specific binding of the VDR to a vitamin D sterol response element (VDRE).11 The DNA-binding domain of the VDR contacts the major groove of a double-stranded hexameric DNA sequence with the consensus motif RGKTCA (R=A or G, K=G or T). In most cases the heterodimeric partner of VDR is the retinoid X receptor (RXR), another nuclear receptor superfamily member, which also contacts DNA.12 Simple VDREs are often formed by a direct repeat of two hexameric core binding motifs spaced by three nucleotides (DR3-type VDRE). This type of VDRE13 has been reported in the proximal promoter of a number of 1α,25(OH)2D3 responding genes including the human vitamin D3 24-hydroxylase (CYP24) and the rat atrial natriuretic factor (ANF) gene.14, 15

Ligand binding to the VDR causes a conformational change within its complete ligand-binding domain (formed by 12 α-helices). This results in the replacement of the corepressor protein responsible for mediating unliganded VDR repression by coactivator proteins of the p160-family.16 These coactivators link the ligand-activated VDR to enzymes displaying histone acetyltransferase activity, such as cAMP response element-binding (CREB) binding protein (CBP), that cause chromatin relaxation and thereby reversing the action of unliganded VDR.17 In a subsequent step, ligand-activated VDR changes rapidly from interacting with the coactivators of the p160-family to those of mediator complexes, which form a bridge to the basal transcriptional machinery with RNA polymerase II (pol II) at its core. In this way ligand-activated VDR executes two tasks, the modification of chromatin and the regulation of transcription.

Many of the natural ligands of nuclear receptors are linked in the way that they are metabolic products of cholesterol.1 It is therefore not surprising that many of the primary target genes of these nuclear receptors are the enzymes, such as CYP24, which regulate the levels of intermediate and active cholesterol derivatives. The CYP24 gene is the most responsive primary 1α,25(OH)2D3 target gene and shows at the mRNA level a more than 10,000-fold inducibility by the hormone.18 In contrast, most other known primary 1α,25(OH)2D3 target genes appear to be much less responsive and often show an inducibility of twofold or less after short-term treatment with 1α,25(OH)2D3.19, 20 One of these genes, cyclin C,21 is a functional part of mediator protein complexes involved in gene repression.22 This example highlights the fact that a subset of these genes are involved in transcriptional regulation. This group does not only contain coregulators,23 but also some transcription factors including other nuclear receptors.2 Thus, through different levels, nuclear receptors can have a strong influence over each other's activity. Since PPARδ is the only PPAR subtype that is co-expressed with VDR in many cell types, we investigated whether activation of either of these nuclear receptors had any effect on each other's ability to induce gene expression.

Here, we demonstrate that the moderately expressed human PPARδ gene is a primary 1α,25(OH)2D3-responding gene. In silico analysis of the human PPARδ promoter revealed a DR3-type VDRE approximately 350 bp upstream of the transcription start site (TSS) that was able to bind VDR-RXR heterodimers and upregulate reporter gene activity. Chromatin immuno-precipitation (ChIP) assays demonstrated that VDR, RXR and pol II show a 1α,25(OH)2D3-dependent association with a region of the proximal PPARδ promoter that contains the putative DR3-type VDRE. This was also the case for other proteins that are involved in or subject of chromatin modification, such as CBP and histone 4, which displayed ligand-dependent association and acetylation, respectively. Moreover, 1α,25(OH)2D3 and the synthetic PPARδ ligand L783483 interfere time-dependently in each others effects on VDR mRNA expression, so that their combined application shows complex effects on the induction of the VDR target gene CYP24. This suggests that VDR and PPARδ signaling pathways are interconnected by the reciprocal effects of the activated receptors.

Section snippets

Basal expression of PPARδ mRNA in comparison to cyclin C and CYP24 in different cancer cell lines

The basal mRNA expression level of the PPARδ gene, the VDR gene and two primary 1α,25(OH)2D3 target genes, cyclin C21 and CYP24,24 were monitored by real-time quantitative PCR in relation to the control gene acidic riboprotein P0 (ARP0, also known as 36B4) in MCF-7 and MDA-MB453 human breast cancer cells and in LNCaP and PC-3 human prostate cancer cells (Figure 1(a)). MCF-7 and LNCaP are less agressive, estrogen and testosterone-dependent cell lines, respectively, while MDA-MD453 and PC-3 are

Discussion

Here, we report the human PPARδ gene as a primary 1α,25(OH)2D3 target and demonstrate that PPARδ activation affects VDR-mediated signaling. Both PPARδ and VDR are widely expressed and our observation suggests the interaction of the endocrine signaling pathways of both receptors in numerous tissues. The observation that the PPARδ gene is a 1α,25(OH)2D3-responding gene is interesting, since its function is not linked to the classical endocrine functions of 1α,25(OH)2D3, such as the regulation of

Cell culture

MCF-7, MBA-MD453 (human breast cancer), LNCaP and PC-3 (human prostate cancer) cell lines were grown in phenol red-free Dulbecco's modified eagle's medium (DMEM) and RPMI, respectively, supplemented with 5% (v/v) charcoal-treated fetal bovine serum, 2 mM l-glutamine, 0.1 mg/ml of streptomycin and 100 units/ml of penicillin, in a humidified 95% air/5% CO2 incubator. Prior to mRNA extraction or ChIP assay the cells were treated at a density of 50–60% confluency for the indicated periods and

Acknowledgements

We thank Drs Lise Binderup and Mogens Madsen for 1α,25(OH)2D3 and L783483, and Marjo Malinen and Maija Hiltunen for help with cell culture. Grants from the Academy of Finland, the Finnish National Technology Organization TEKES and the Finnish Cancer Organization supported this research. The authors declare that they have no conflict of interest.

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