Retinoic acid modulates retinaldehyde dehydrogenase 1 gene expression through the induction of GADD153–C/EBPβ interaction
Introduction
Retinoic acid (RA) is a metabolite of vitamin A (retinol) that regulates developmental pathways in vertebrate animals. It exerts its effects through two families of nuclear receptors, RA receptors (RARs) and retinoid X receptors (RXRs). Maintenance of RA homeostasis is crucial for normal embryogenesis and development in chordate organisms.
RA synthesis involves the oxidation of retinol to retinal and the subsequent oxidation of retinal to RA by members of the aldehyde dehydrogenase family (ALDH) [1]. ALDHs are a group of NADP-dependent enzymes that catalyze the conversion of aldehydes into acid metabolites. They also participate in the metabolism of alcohols [2], biogenic amines [3], vitamins [4], steroids [5] and lipids [6], [7], as well as in the biotransformation of numerous drugs and environmental agents [8].
To date, 555 ALDH genes have been described, including 172 in eukaryotes, and 17 functional genes in humans [9]. Among them, cytosolic human ALDH1, mouse retinaldehyde dehydrogenase (Raldh) 1 and mouse Raldh2 have been shown to mediate retinaldehyde oxidation and are considered to play a major role in RA synthesis [10]. A critical role for Raldh2 in mammalian development was established by the finding that embryos homozygous null for Raldh2 failed to develop due to severe abnormalities [11]. On the other hand, Raldh1 null mouse presents an insulin resistance and increased energy dissipation [12].
The mouse Raldh1 gene has similar tissue-specificity and developmental control as the human ALDH1 gene. It is highly expressed in adult mouse tissues, including liver [13], [14], and plays a role in RA clearance in vivo[15]. Although the mouse Raldh1 gene has been sequenced and the promoter region has been characterized, little is known about the molecular mechanisms by which expression of this gene is regulated.
Recently, it has been shown that the aryl hydrocarbon receptor (AhR) is involved in the RA metabolism. The AhR is a ligand activated receptor [16], and a member of the basic helix–loop–helix-PAS (bHLH-Per-Arnt-Sim) transcription factor family. Upon binding ligand, the AhR translocates to the nucleus, and up-regulates the expression of a battery of genes encoding xenobiotic-metabolizing enzymes, such as cytochrome P450s (CYP1A1, CYP1A2, CYP1B1), NAD(P)H quinone oxydoreductase and UDP-glucoronosyl-transferase-6 [17]. Livers from AhR-null mice show a 3-fold increase in RA levels as well as a down regulation of Raldh1 and 2, suggesting that AhR controls RA catabolism [18]. More recently, it was determined that expression of CYP2C39, a mouse enzyme responsible for RA 4-hydroxylation, is under AhR control also [19]. These data explain the high levels of RA observed in AhR-null mouse liver. Because AhR-null mouse has a defect in RA catabolism leading to elevated RA levels and decreased Raldh1 mRNA levels, this mouse model is an important tool to study the role of RA on Raldh1 gene regulation.
We previously observed that RA mediates feedback inhibition of the human ALDH1 gene expression through decreasing C/EBPβ binding to CCAAT box response element located at the ALDH1 promoter [20]. However, the molecular mechanism of this inhibition is still unknown. In order to determine the molecular nature of this effect, we first investigated whether the mouse Raldh1 gene expression is regulated by a similar mechanism as determined for the ALDH1. As in human ALDH1, RARα and C/EBPβ bind to a novel RA response element (RARE) and to the CCAAT box, respectively, located within the Raldh1 promoter. We also show that treatment with RA increased GADD153 mRNA and GADD153–C/EBPβ interaction resulting in a sequestration of C/EBPβ and decreased C/EBPβ binding to the Raldh1 CCAAT box.
Section snippets
Materials
Mouse hepatoma-derived cells (Hepa-1) were obtained from ATCC (Manassas, VA, USA). All-trans-RA was obtained from Sigma (St. Louis, MO). RARα and RXRβ antibodies were purchased from Affinity Bioreagents (Golden, CO). Raldh1 cDNA was provided by Gregg Duester (Burnham Institute, La Jolla, CA) [21] and the type II transglutaminase (TG-II) and RARα cDNA were provided by Ronald M. Evans (Howard Hughes Medical Institute, San Diego, CA) [22].
Cell culture
Hepa-1 cells were cultured in Dulbecco's modified Eagle's
Results
In order to confirm the alteration on RA metabolism observed in AhR-null mice, we first evaluated the expression of hepatic genes under RA control. Transglutaminase 2 (TG-II) is an inducible transamidating acyltransferase that catalizes Ca2+-dependent protein modification. Its expression is up-regulated by RA [30]. Fig. 1 shows that AhR-null mouse liver presents a 5-fold increase on TG-II mRNA levels compared with wild-type mice. These results are in agreement with previous reports describing
Discussion
RA has marked effects on cell differentiation and proliferation. Since oxidation of retinaldehyde to RA is irreversible, this reaction must be tightly regulated. Therefore, understanding the molecular mechanisms controlling RA synthesis is of great importance. The present study demonstrated that RARα and C/EBPβ bind to the mouse Raldh1 gene 5′-flanking region and that this interaction resembles that reported for human ALDH1 gene regulation [20]. The mouse Raldh1 −75/−18 bp regions is highly
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