TCDD administration after the pro-adipogenic differentiation stimulus inhibits PPARγ through a MEK-dependent process but less effectively suppresses adipogenesis

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Abstract

Hormone (IDMB)-induced adipogenesis in C3H10T1/2 cells is suppressed by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the aryl hydrocarbon receptor (AhR). We have previously reported that TCDD addition 48 h before the hormonal stimulation of IDMB suppresses a key mediator of adipogenesis, the peroxisome proliferator-activated receptor (PPARγ), by a MEK/ERK dependent mechanism. Here we add to previous evidence that this synergism functions after IDMB addition but before increased PPARγ1 transcription. Suppression remains effective and MEK/ERK dependent when TCDD is added 6–12 h after IDMB addition but not when delayed to 16–24 h, thus preceding the rise in PPARγ mRNA. TCDD suppression of the number of committed adipocytes and of triglyceride formation is less effective with the delayed addition. TCDD therefore does not directly suppress the expression of the key mediator PPARγ1. An alternative mediation of adipocyte commitment is apparently less sensitive to the 6–12 h of delayed TCDD addition. TCDD suppression potencies (EC50 = 50 pM) match the potencies for stimulation of CYP1B1 protein and AhR-sensitive reporters. The AhR antagonist 3′-methoxy-4′-nitroflavone (3-MNF) inhibited both TCDD-mediated CYP1B1 induction and inhibition of PPARγ protein expression. This antagonism was only effective when 3-MNF was present in the 24-h period after IDMB addition. TCDD activation of AhR in conjunction with MEK/ERK therefore generates PPARγ1 suppression activity before the increase of PPARγ1 synthesis. The potency and inhibition data are consistent with induction of one or more gene products that sustain suppression through the extended period of PPARγ1 transcription.

Introduction

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) arises from chlorination of phenolic substrates (Schmidt and Bradfield, 1996) or from partial combustion of organic materials in the presence of chlorine sources (Remillard and Bunce, 2002). TCDD is the prototypical agonist of the aryl hydrocarbon receptor (AhR), an orphan nuclear receptor that is a basic helix–loop–helix PAS protein (Schmidt and Bradfield, 1996). Concern over TCDD as a risk factor in humans is highlighted by its extraordinarily slow metabolism and consequent lifetime accumulation in body fat Flesch-Janys et al., 1996, Weiss et al., 2003, Wolfe et al., 1994. Adipose tissue is a target organ for insulin activity and is significant in maintaining glucose homeostasis (Matthaei et al., 2000). Recent epidemiological studies suggest a link between human exposure to dioxins and the development of Type II diabetes Longnecker and Michalek, 2000, Michalek et al., 1999, Steenland et al., 2001. A linkage of TCDD exposure to impaired insulin-dependent glucose response has been seen in mice (Liu and Matsumura, 1995), and a much stronger correlation of TCDD blood levels with insulin-dependent glucose tolerance has been identified (Remillard and Bunce, 2002). From these reports, it is therefore tempting to hypothesize that activation of AhR can lead to aberrant effects on glucose homeostasis and contribute to insulin resistance.

Type II diabetes is characterized by a failure of insulin to stimulate the normal uptake of blood glucose into muscle and fat cells. Chronic attenuation of insulin-induced glucose uptake may combine with other susceptibility factors to produce Type II diabetes. An effective clinical treatment of Type II Diabetes has been the thiazolidinediones, a class of drugs that enhance glucose uptake and have been shown to activate the nuclear transcription factor peroxisome proliferator-activated receptor (PPARγ), a central mediator of the adipogenic program (Spiegelman, 1998). Other evidence that links Type II diabetes and PPARγ has been reported. Mutations in PPARγ cause abrogated glucose tolerance and Type II diabetes in human subjects (Auwerx, 1999) and have also been documented to correlate with increased insulin sensitivity, decreased body mass index, and improved lipid profile (Picard and Auwerx, 2002).

In vitro models used to study adipogenesis include murine multipotential embryonic fibroblasts, which can be transformed into fat, muscle, or bone under the appropriate stimulation (Taylor and Jones, 1979). The mouse 3T3-L1 cell line provides a model for pre-adipocytes, although the embryo fibroblast C3H10T1/2 cell line provides a model for multipotential differentiation in which only 50% of the population differentiates into adipocytes. Adipogenesis is directed by a tightly regulated cascade of gene expression and is initiated after the stimulus provided by the hormonal cocktail IDM, which consists of insulin (I), the glucocorticoid dexamethasone (D), and a means of cAMP stimulation (isobutylmethylxanthine; M) Cornelius et al., 1994, Gregoire et al., 1998. This differentiation process is divided into an early phase (days 0–2) and a late phase (days 2–8). During the early phase, cells exit the cell cycle and commit to the differentiation pathway, although in the second phase, the committed cells are stimulated in insulin-only media to elevate the expression of genes involved in triglyceride synthesis.

In part, we are using adipogenic 10T1/2 differentiation as a model to explore how chemicals may disrupt the exit from the cell cycle, which is an essential, early step in differentiation. 10T1/2 cells have also recently been used for a set of studies that demonstrate a similar suppression of adipogenesis by arsenite Trouba et al., 2000, Wauson et al., 2002 and mercuric ions (Barnes et al., 2003). TCDD-mediated AhR signaling blocks adipogenesis in 10T1/2 cells (Alexander et al., 1998) and in 3T3-L1 cells (Phillips et al., 1995). We and others find that 10T1/2 cells differentiate more readily into adipocytes when a PPARγ ligand such as the thiazolidinedione BRL46593 is added with IDM Alexander et al., 1998, Hwang et al., 1997. Hereafter, this adipogenic hormonal cocktail of insulin, dexamethasone, isobutylmethylxanthine, and BRL46593 is referred to as IDMB.

There are two isoforms of PPARγ, PPARγ1 and PPARγ2, that result from activation of separate promoters but involve alternative splicing such that the γ2-isoform is 30 amino acids longer at the N-terminus (Rosen et al., 2000). In 10T1/2, the PPARγ1 isoform peaks in expression between 24 and 48 h after the addition of IDMB, followed by increases of the γ2-isoform in the second phase (Hanlon et al., 2003). The PPARγ isoforms also mediate the insulin-dependent induction of lipogenic genes such as aP2 and Glut4 (Spiegelman, 1998). PPARγ has also been shown to direct the synthesis of triglycerides, which appear as lipid droplets (Chawla and Lazar, 1994).

TCDD treatment decreases PPARγ1 protein expression in close parallel to decreases in the subsequent insulin-stimulated changes in these and other lipogenic genes Alexander et al., 1998, Hanlon et al., 2003, Liu et al., 1998. In our studies, we have focused on the TCDD inhibition of PPARγ protein expression at 48 h in relation to effects on insulin-dependent adipogenesis 6 days later. We have recently demonstrated that TCDD added 48 h before hormonal stimulation suppresses PPARγ protein expression only in conjunction with activation of growth factor receptors that stimulate MEK and ERK kinase activities (Hanlon et al., 2003). This suppression is lost when serum-induced ERK activation is removed but is restored by EGF or transfection of constitutively active MEK (Hanlon et al., 2003). However, TCDD did not affect the extent of ERK activation. We have also found that this activity is restricted to a limited time window in the early differentiation process, which occurs after the peak stimulation of C/EBPβ and before the onset of PPARγ expression (Hanlon et al., 2003). This TCDD-mediated inhibition of PPARγ and subsequent adipogenesis are equally alleviated when the MEK inhibitors PD98059 or U0126 are present either at the time of IDMB initiation or when added 6–12 h later. These inhibitors are completely ineffective when added after 16–24 h. These data support the concept that activation of AhR works in concert with ERK activation to affect genes that are expressed before PPARγ1.

In this study, we have set out to further characterize the period of TCDD-activated AhR activity in relation to TCDD inhibition of PPARγ and adipogenesis. Previous work in 3T3-L1 cells has indicated that TCDD inhibition of lipid-accumulating cells remains effective if TCDD was added after the hormonal stimulation (Phillips et al., 1995). Here we have administered TCDD after IDMB stimulation to further define the period of TCDD inhibition of PPARγ. In particular, we are interested in whether this coincides with the time frame of effective EGF/MEK signaling and secondly whether this delayed addition retains dependence on MEK. We have looked at several end points that are affected at different times. These include PPARγ expression changes at 20–32 h, the proportion of adipocytes, and the total generation of triglycerides.

These analyses show that TCDD addition after IDMB stimulation is appreciably less effective than pretreatment in suppressing mature adipocyte formation but retains full effectiveness in suppressing PPARγ1 mRNA and protein. This discrepancy is discussed in terms of early pro-adipogenic genes that are induced before PPARγ1 that may be less sensitive to delayed TCDD addition.

Section snippets

Cell culture

10T1/2 cells (American Type Culture Collection, Bethesda, MD) were grown in Dulbecco's Modified Eagle Medium/F-12 (DMEM/F-12) nutrient mixture (Gibco/Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA), 75 units of penicillin, and 75 μg/μl streptomycin. Cells were maintained at 37 °C in a humidified CO2 incubator. The differentiation of these cells was carried out as follows. At 100% confluency, cells were administered DMEM/F-12

TCDD suppression of adipogenesis and PPARγ synthesis depends on the time of addition

Previous work in 3T3-L1 cells has shown that the effect of TCDD on adipogenesis declines as the pre-incubation time decreases but is maintained when TCDD is added after hormonal initiation (Phillips et al., 1995). We have used selective MEK inhibitors to show that this MAP kinase pathway functions synergistically with TCDD in a relatively narrow time window that starts 6 h after hormonal initiation. Here we have set out to define the window of effectiveness of TCDD and to test whether

Discussion

In this study, we have further characterized TCDD-mediated inhibition of PPARγ and adipogenesis. We demonstrate that TCDD addition 6–12 h after IDMB is as effective in lowering PPARγ expression as a TCDD administration 48 h before hormonal stimulation. We also show that a limit in this effective period is reached at about 16 h after IDMB administration. Thus, the period for effective TCDD suppression precedes the major onset of PPARγ1 induction at 18–20 h (Hanlon et al., 2003). This provides

Acknowledgements

This publication was made possible by NIH grant R01 DK55302 and National Institute of Environmental Health Sciences (NIEHS) grant number T32 ES07015. MAC is supported by the National Institute of Diabetes and Digestive and Kidney Diseases grant number F31 DK62525. The authors thank Daniel J. Felkner and Dr. Renata Jaskula-Sztul for technical assistance and Drs. Thomas Gasiewicz and Ellen C. Henry for reagents. The authors also wish to thank Dr. Emery H. Bresnick for access to the ABI 7000

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