Microarray analysis of early adipogenesis in C3H10T1/2 cells: Cooperative inhibitory effects of growth factors and 2,3,7,8-tetrachlorodibenzo-p-dioxin

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Abstract

C3H10T1/2 mouse embryo fibroblasts differentiate into adipocytes when stimulated by a standard hormonal mixture (IDMB). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), via the aryl hydrocarbon receptor (AhR), inhibits induction of the key adipogenic gene peroxisome proliferator-activated receptor γ (PPARγ) and subsequent adipogenesis. This TCDD-mediated inhibition requires activation of the extracellular signal-regulated kinase (ERK) pathway, which can be accomplished by serum, epidermal growth factor (EGF), or fibroblast growth factor (FGF). In the absence of serum or growth factors, IDMB induced adipogenesis without mitosis. Microarray analysis identified 200 genes that exhibited expression changes of at least twofold after 24 h of IDMB treatment. This time precedes most PPARγ stimulation but follows the period of TCDD/ERK cooperation and periods of increased cell contraction and DNA synthesis. Functionally related gene clusters include genes associated with cell structure, triglyceride and cholesterol metabolism, oxidative regulation, and secreted proteins. In the absence of growth factors TCDD inhibited 30% of these IDMB responses without inhibiting the process of differentiation. A combination of EGF and TCDD that blocks differentiation cooperatively blocked a further 44 IDMB-responsive genes, most of which have functional links to differentiation, including PPARγ. Cell cycle regulators that are stimulated by EGF were substantially inhibited by IDMB but these responses were unaffected by TCDD. By contrast, TCDD and EGF cooperatively reversed IDMB-induced changes in cell adhesion complexes immediately prior to increases in PPARγ1 expression. Changes in adhesion-linked signaling may play a key role in TCDD affects on differentiation.

Introduction

A variety of immortalized fibroblast cell lines of murine origin, including the multipotential C3H10T1/2 (10T1/2) cell line, can be induced to differentiate into adipocytes with a mixture (IDM) of insulin, isobutylmethylxanthine, and dexamethasone. Adipogenesis requires the IDM-mediated, sequential activation of multiple genes and gene families, including peroxisome proliferator-activated receptor (PPAR)γ, that have previously been shown to be both required and sufficient for adipocyte differentiation in vivo and in vitro (Gregoire et al., 1998, Rosen and Spiegelman, 2000). An activator of the PPARγ receptor, such as BRL49653 (BRL), enhances insulin-dependent lipogenesis in 10T1/2 cells (Paulik and Lenhard, 1997).

The ubiquitous environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits both adipogenesis (Alexander et al., 1998, Brodie et al., 1997, Phillips et al., 1995) and the function of mature adipocytes (Kern et al., 2002a, Kern et al., 2002b, Nagashima and Matsumura, 2002). TCDD binds to the aryl hydrocarbon receptor (AhR), which then becomes activated to mediate transcription of a limited set of genes containing the specific DNA recognition sequence (Probst et al., 1993). The effect of TCDD on IDMB-stimulated adipogenesis in 10T1/2 cells correlates with a suppression of the large elevation of PPARγ expression and is maximally effective when TCDD is added 48 h prior to IDMB stimulation (Cimafranca et al., 2004). Furthermore, TCDD-mediated inhibition of adipogenesis depends on activation of the mitogen/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway during a period 6–20 h after the initiation of differentiation, which precedes the onset of PPARγ induction (Cimafranca et al., 2004, Hanlon et al., 2003). This paper extends previous research to provide a strong molecular basis for adverse effects of TCDD on PPARγ expression and adipogenesis (Alexander et al., 1998, Cimafranca et al., 2004, Hanlon et al., 2003).

Growth factors present in serum, such as fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), are potent activators of signaling pathways including the ERK pathway as well as other kinase pathways (Heasley and Johnson, 1992, Marshall, 1995, Zheng et al., 1999). Growth factors have also been shown to have effects on adipocyte differentiation (Adachi et al., 2001, Boone et al., 2000, Hauner et al., 1995, Krieger-Brauer and Kather, 1995, Prusty et al., 2002, Tabata et al., 2000) and mature adipocytes (Garcia and Obregon, 2002, Gould et al., 1994, Hauner et al., 1995, Van Epps-Fung et al., 1996). We have recently shown that 10T1/2 cells do not require mitosis for differentiation and therefore readily differentiate in absence of any external mitogen, such as serum renewal, growth factors, or insulin (Cho and Jefcoate, 2004). There is therefore no requirement for MEK/ERK to produce a mitotic change in 10T1/2 cells. Thus, EGF alone does not affect differentiation but can provide the ERK activation required for TCDD to inhibit adipogenesis (Hanlon et al., 2003).

Several recent microarray studies have investigated the time-dependent regulation of gene expression during adipocyte differentiation (Burton et al., 2002, Guo and Liao, 2000, Ross et al., 2002). In 3T3-L1 cells, the differentiation stimulus produces sequential waves of gene expression (Ntambi and Young-Cheul, 2000). Array studies have also addressed in vitro gene expression in comparison to expression in stromal cells and adipocytes in vivo (Ross et al., 2002, Soukas et al., 2001). Here we describe the use of microarrays in C3H10T1/2 cells to document mRNA profiles at a single time (24 h of stimulation) that represents an early transition between distinct phases of the differentiation process. Importantly, this time is based on the time requirements for TCDD and MEK/ERK activity (Hanlon et al., 2003). We hypothesize that the cooperation of the AhR/TCDD and ERK signaling pathways alters the expression of an intermediate cluster of genes that define an early differentiation pathway leading to induction of PPARγ1.

This work also examines gene expression during differentiation in absence of cell division provided by omission of the mitogenic serum renewal (Hanlon et al., 2003). We compare these profiles to those provided by an additional EGF mitotic stimulus that does not affect differentiation. We show that a substantial set of cell cycle genes is activated by EGF but are inhibited by the adipogenic stimulus and unaffected by TCDD. We have used the multiple conditions involving the selective effects of TCDD and EGF on differentiation to provide a distinctive assessment of functionally important gene expression during this early stage of the differentiation process. This is essential to understanding the mechanisms of disruption generated by the synergism between AhR and growth factor/ERK signaling. Importantly, we find that most inhibition of IDMB-induced gene responses by TCDD occurs without inhibition of differentiation, and that cooperative inhibition of differentiation by TCDD and EGF corresponds to an effect on only 44 genes. Also, EGF and TCDD inhibit a distinct set of IDMB-regulated genes to similar extents under adipogenic conditions. We provide evidence that this remarkable association may arise from effects of TCDD on cell adhesion and the associated signaling.

Section snippets

Materials

TCDD in toluene was purchased from Chemsyn Science Laboratories (Lenexa, KS). Tissue culture plates were from Fisher Chemical Company (Pittsburgh, PA). Materials for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) were obtained from Bio-Rad Laboratories (Hercules, CA). BCA-Protein reagents were purchased from Pierce. Nitrocellulose membranes, enhanced chemiluminescence (ECL) detection kit, and hyperfilm–ECL luminescence detection films were obtained from Amersham

EGF and FGF restore TCDD sensitivity to the unrenewed serum protocol

PPARγ occurs in two forms, PPARγ1 and PPARγ2, that are derived from different promoters of the same gene (Zhu et al., 1995). In 10T1/2 cells, PPARγ1 expression peaks within 48 h after IDMB stimulation, whereas PPARγ2 expression increases later (Hanlon et al., 2003). Here we aim to test whether the AhR can play a role in the effects of EGF and FGF on IDMB-induced PPARγ1 expression and subsequent differentiation. PPARγ expression and adipocyte differentiation were fully stimulated without the

Discussion

The data presented here demonstrate that the growth factors EGF and FGF function synergistically with TCDD-mediated activation of the AhR to suppress both IDMB-induced PPARγ expression and adipocyte differentiation (Fig. 1). We have previously shown that ERK activity is essential to TCDD-mediated suppression (Hanlon et al., 2003). Differentiation is maintained in these 10T1/2 cells without the serum renewal (URS protocol) that typically accompanies hormonal stimulation and which activates the

Acknowledgments

This publication was made possible by NIH grants R01 DK55302, DK062525, ES09090, and training grant number T32 ES07015 from the National Institute of Environmental Health Sciences (NIEHS), NIH (Contribution # 360, Molecular and Environmental Toxicology Center).

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