Peroxisome proliferator-activated receptorα agonists differentially regulate inhibitor of DNA binding expression in rodents and human cells

Abstract

Inhibitor of DNA binding (Id2) is a helix-loop-helix (HLH) transcription factor that participates in cell differentiation and proliferation. Id2 has been linked to the development of cardiovascular diseases since thiazolidinediones, antidiabetic agents and peroxisome proliferator-activated receptor (PPAR) gamma agonists, have been reported to diminish Id2 expression in human cells. We hypothesized that PPARα activators may also alter Id2 expression. Fenofibrate diminished hepatic Id2 expression in both late pregnant and unmated rats. In 24 hour fasted rats, Id2 expression was decreased under conditions known to activate PPARα. In order to determine whether the fibrate effects were mediated by PPARα, wild-type mice and PPARα-null mice were treated with Wy-14,643 (WY). WY reduced Id2 expression in wild-type mice without an effect in PPARα-null mice. In contrast, fenofibrate induced Id2 expression after 24 hours of treatment in human hepatocarcinoma cells (HepG2). MK-886, a PPARα antagonist, did not block fenofibrate-induced activation of Id2 expression, suggesting a PPARα-independent effect was involved. These findings confirm that Id2 is a gene responsive to PPARα agonists. Like other genes (apolipoprotein A-I, apolipoprotein A-V), the opposite directional transcriptional effect in rodents and a human cell line further emphasizes that PPARα agonists have different effects in rodents and humans.

Figure 1

Fenofibrate downregulates Id2 gene expression in rats. Left panel: relative amount of mRNA of liver Id2, after 4-day treatment with or without fenofibrate in virgin and pregnant rats, measured by semiquantitative RT-PCR. Values were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and were represented using arbitrary units. Capital letters correspond to the statistical comparisons between pregnant and virgin rats receiving the same treatment. Small letters correspond to the statistical comparisons between rats receiving different drug doses. Values not sharing a common letter are significantly different at P < 0.05. Each value represents the mean ± standard error of five animals. Right panel: starvation downregulates Id2 gene expression. Relative amount of mRNA of liver Id2 from pregnant rats fed with standard pellet or fasted 24 h, measured by semiquantitative RT-PCR. Values were normalized against GAPDH expression and were represented using arbitrary units. Asterisk represents significantly different at P < 0.05.

Figure 2

Downregulation of Id2 gene expression by WY is dependent on PPARα. Wild-type SV129 mice (+/+) or SV129 mice that lack PPARα (−/−) were fed a control diet (Control) or a diet containing WY (0.1%) or DEHP (0.6%) for 3 weeks. Total RNA isolated from liver was separated by 1.2% agarose, transferred to nylon, and analysed by northern blot using probes for Id2, Ehhadh, and 17βHSD, and GAPDH as a control. Northern autoradiograms (a) were densitometrically scanned and expression normalized to that of GAPDH (b). Each value represents the mean ± standard error of three animals. *significantly different from control (P < 0.05).

Figure 3

Fenofibrate upregulates Id2 gene expression in HepG2 cells. Panel (a) human hepatocarcinoma cells treated with different concentrations of fenofibrate (0, 10, 50, or 100 μM) for 2, 6, or 24 hours. Relative Id2 mRNA levels were measured by real-time PCR, normalized to β-actin levels and expressed in relative units to control. Values for Id2 mRNA are expressed as mean ± SD (n = 3). Panel (b) HepG2 cells were preincubated with the PPARα antagonist MK-886 (10 μM) where indicated and treated with different concentrations of fenofibrate (0 or 50 μM) for 24 hours.