Activation of PPARγ is not involved in butyrate-induced epithelial cell differentiation

Abstract

Histone deacetylase-inhibitors affect growth and differentiation of intestinal epithelial cells by inducing expression of several transcription factors, e.g. Peroxisome proliferator-activated receptor γ (PPARγ) or vitamin D receptor (VDR). While activation of VDR by butyrate mainly seems to be responsible for cellular differentiation, the activation of PPARγ in intestinal cells remains to be elucidated. The aim of this study was to determine the role of PPARγ in butyrate-induced cell growth inhibition and differentiation induction in Caco-2 cells. Treatment with PPARγ ligands ciglitazone and BADGE (bisphenol A diglycidyl) enhanced butyrate-induced cell growth inhibition in a dose- and time-dependent manner, whereas cell differentiation was unaffected after treatment with PPARγ ligands rosiglitazone and MCC-555. Experiments were further performed in dominant-negative PPARγ mutant cells leading to an increase in cell growth whereas butyrate-induced cell differentiation was again unaffected. The present study clearly demonstrated that PPARγ is involved in butyrate-induced inhibition of cell growth, but seems not to play an essential role in butyrate-induced cell differentiation.

Introduction

Although data reported in humans are still controversial [1], evidence has been provided that alimentary fibers exert a protective effect against colon carcinogenesis in rodents [2], [3], [4]. The protective effect is linked to the capacity of fibers to be metabolized into short chain fatty acids as butyrate [5]. Whereas in normal epithelial cells butyrate is the preferred oxidative fuel and stimulates growth [6], [7], it has been shown to inhibit proliferation and induce rapid cell differentiation in colon carcinoma cell lines [7], [8], [9].

Peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors that belong to the nuclear hormone receptor superfamily, also including the receptors for thyroid hormone, retinoids, steroid hormones and vitamin D [10], [11]. They occur in three different isotypes termed α, β (also called δ, NUC-1 or FAAR) and γ. Moreover, in man, three PPARγ-mRNA isoforms have been described, PPARγ1, γ2 and γ3 [12]. Upon activation, PPARs heterodimerize with another nuclear receptor, the 9-cis-retinoid X receptor (RXR), and alter the transcription of numerous target genes after binding to a specific DNA binding site termed PPRE (peroxisome proliferator responsive element) [13], [14]. It has been demonstrated that 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2), a metabolite of prostaglandin D2, is a potential endogenous ligand for PPARγ [15], and that thiazolidinediones (synthetic antidiabetic agents) such as troglitazone, rosiglitazone, ciglitazone and MCC-555 are specific exogenous ligands for PPARγ [16]. Other PPARγ-ligands has been described, including eicosanoids [17] and non-steroidal anti-inflammatory drugs [18].

PPARγ is expressed at high levels in colonic epithelial cells and colon cancer cells [19], [20]. Sarraf and colleagues [21] showed that activators of PPARγ suppress the growth response of colon cancer cells. Brockmann and colleagues reported that activation of PPARγ leads to inhibition of anchorage independent growth of colon cancer cells [22]. On the other hand, Seaz and colleagues [23] and Lefebvre and colleagues [24] showed in APC^MIN (adenomatous polyposis coli) mice, an animal model of familial polyposis, that treatment with PPARγ ligands increases the frequency and size of colon polyps. Although the results of these in vitro and in vivo studies are contradictory, it is very likely that the PPARγ pathway can modulate the growth response of colon epithelial cells and colon cancer cells.

On the basis of our published finding, that NaB (sodium butyrate) upregulates PPARγ in colon cancer cells [25], this study is addressed to elucidate the role of PPARγ in butyrate-induced differentiation and/or growth response.