Identification and characterization of a novel mouse peroxisome proliferator-activated receptor a-regulated and starvation-induced gene, Ppsig

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Abstract

The peroxisome proliferator-activated receptor alpha (PPARa) has been known to play a pivotal role in maintaining the energy balance during fasting; however, the battery of PPARa target genes involved in this metabolic response is still not fully characterized. Here, we report the identification and characterization of Ppsig (for PPARa-regulated and starvation-induced gene) with unknown biological function from mouse liver. Multiple Ppsig cDNAs which differed in the 3'-untranslated regions were identified. The open reading frame of Ppsig cDNA is 1830 bp which encodes a protein of 67.33 kDa. Ppsig contains 11 exons spanning at least 10 kb. Although the exact biological function of Ppsig is still not known, we found that Ppsig mRNA transcript was dramatically up-regulated during 72 h fasting and following treatment with a potent PPARa agonist, in a tissue-specific and PPARa-dependent manner. A functional peroxisome proliferator-response element was found in the intron 1 of Ppsig, thus confirming that Ppsig is a novel direct mouse PPARa target gene. This finding might help in elucidating the transcriptional regulatory mechanism of Ppsig in the cellular response to fasting.

Introduction

The peroxisome proliferator-activated receptor alpha (PPARa), a nuclear hormone receptor and a ligand-dependent transcriptional factor, has been known to play a key role in many biological processes such as lipid metabolism (Lee, Kim, Zhao, Cha, & Kim, 2004), amino acid metabolism (Kersten et al., 2001) and glycogen metabolism (Mandard et al., 2007). It is known that PPARa mediates its action via modulations of its target gene expressions. PPARa regulates its target genes by forming heterodimer with retinoid X receptor alpha (RXRa) and binds to a DNA consensus sequence named as the peroxisome proliferator-response element (PPRE) on its target genes (Desvergne & Wahli, 1999). The first PPRE was identified in the 5'-flanking of rat acyl-CoA oxidase gene (Tugwood et al., 1992). Since then many PPREs have been reported in the proximal promoters of a large number of PPARa target genes (Mandard, Muller, & Kersten, 2004). It is anticipated that more new PPARa target genes are awaited to be discovered.

As part of our continued efforts in identifying novel PPARa target genes in response to the metabolic perturbation during fasting, we have used the fluorescent differential display (FDD) to compare the liver mRNA profiles of wild-type (WT) and PPARa-null (KO) mice under both fed and fasted conditions, and to isolate PPARa-dependent and fasting-responsive genes (Lee, Tian, Lee, & Cheung, 2002). In the present paper, we describe the isolation, cloning and characterization of a novel PPARa target gene named Ppsig (for PPARa-regulated and starvation-induced gene) from mouse liver. The exact biological function of Ppsig is not known. But we observed that Ppsig mRNA transcript was dramatically induced in organs involved in lipid metabolism in the WT mice during 72 h fasting. No such induction was observed in the fasted KO mice, suggesting that PPARa is required for the transcriptional up-regulation of Ppsig during fasting. Indeed a functional PPRE was found in the intron 1 of Ppsig, thus confirming that Ppsig is a novel PPARa target gene which might play a significant role in the cellular response during energy deprivation.

Section snippets

Animal and treatment

Male WT and KO mice were used (Lee et al., 1995). For the starvation experiment, fasted animals were deprived of food for 72 h prior to collection of tissue samples. For the Wy-14,643 [(4-chloro-6-(2,3-xylidino)-2-pyrimidinyl-thio)acetic acid] experiment, animals were provided with a 0.1% (w/w) Wy-14,643 (Chemsyn Laboratories, Lenexa, KS) rodent chow diet (Bioserv, Frenchtown, NJ) for 2 weeks ad libitum.

Fluorescent differential display

FDD was used to compare the mRNA expression profiles between the WT and KO mice under both

Identification of the mouse Ppsig by mRNA differential display

To search for novel PPARa target genes involved in the metabolic response to fasting, we compared the liver mRNA expression profiles between the WT and KO mice under both fed and fasted conditions using the fluorescent differential mRNA display and isolated a 570 bp cDNA fragment (Fig. 1A) with unknown biological function. This cDNA fragment was induced in the WT fasted group but not in KO group under the same condition, suggesting that the transcriptional induction of this gene was regulated by

Concluding remarks

In this study, we have cloned and characterized the full-length mouse liver Ppsig cDNA and its genomic sequences. We found that Ppsig mRNA transcript was abundantly expressed in organs with high rate of energy oxidation and its level was transcriptional up-regulated by PPARa in a tissue-specific manner during fasting and treatment with a PPARa agonist. This PPARa-dependent transcriptional up-regulation of Ppsig was further supported by the finding of a functional PPRE in the intron 1 of Ppsig.

Acknowledgements

The work described in this paper was partially supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project Nos. CUHK4241/00M, CUHK4328/03M and 2030275).

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    1

    These authors contributed equally to this work.

    2

    Present address: College of Life Sciences, Shaanxi Normal University, Xi’an 710062, China.

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