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 conserved between human and mouse and it contains an octamer factor binding site, and a CCAAT box. Within this region a putative RARE was found to be located adjacent to the CCAAT box (at –82/–58 bp), and this RARE-like motif was demonstrated to bind the RARα/RXRβ heterodimer by EMSA. Although no apparent RARE consensus sequence was identified within the –82/–58 bp region, this sequence seems to belong to a RA response complex element which has few or no obvious consensus elements. The affinity to the RARα/RXRβ complex shown by this RARE-like motif was similar to that seen with the RARE/DR-5 element, which is one of the most potent inducing sequences found in RAR-responsive element promoters [35
]. RXRβ antiserum super-shifted complex 1, thus indicating that this signal represents the RARα/RXRβ heterodimer. However, a complex 1 signal without shifting was still detected; suggesting that this complex may be also represented by the interaction of RARα with other RXR subtype.
The mouse Raldh2
promoter sequence contains several potential transcription factor-binding sites: CCAAT boxes, AP-1, AP-2, AP-4 and AHR/ARNT. However RARE or RXRE consensus sequences were not predicted [36
]. Nevertheless, after a Raldh2
promoter analysis, we found evidence for a RARE at –2057 bp that is similar to the RARE found in the human ALDH1 and mouse Raldh1 promoters (data not shown). These data suggest that RA may generally control retinal dehydrogenase gene expression through an interaction between RARα and RA response complex elements.
In previous studies we uncovered the molecular mechanism by which RA down regulates ALDH1
gene expression [20
]. This RA inhibitory effect is mediated by decreasing C/EBPβ binding to the promoter. However, the nature of this decrease was unknown. C/EBPβ also binds to the Raldh1
promoter suggesting that Raldh1
transcription may be regulated by this transcription factor. As reported previously, C/EBPβ is a positive regulator of human ALDH1
, and mutation analysis showed that the CCAAT box is critical for its promoter activity. Similarly, Stewart et al. observed that disruption of the CCAAT box in mouse Raldh2
decreased transcriptional activity by 50% [25
]. More recently, the rat Raldh1
promoter was characterized. The authors report a CCAAT box and octamer binding site located between –72 and –68 bp and –56 and –49 bp, respectively, from transcriptional start site. Mutations of CCAAT box abolish transactivation activity, indicating that this motif is critical for basal promoter activity [37
]. These findings suggest that C/EBPβ may generally regulate the expression of retinal oxidizing ALDHs. However, no RA effects were observed on rat Raldh1 mRNA or on promoter activity. The RARE identified in human ALDH1
, and the mouse, and rat Raldh1
promoters were TGTTCA, TGCCCA, and TGGCCA, respectively. These data suggest that T/G change on the putative rat RARE respect with the human is responsible for the lack of responsiveness to RA.
In the present study we found that Hepa-1 cell cultures treated with RA resulted in increased, rather than decreased, C/EBPβ mRNA levels in a time-dependent manner, ruling out that the mechanism by which RA disrupts CCAAT–C/EBPβ interaction is at transcriptional level. Since the Ahr
-null mouse presents elevated hepatic RA concentrations, the finding of increased C/EBPβ mRNA levels in AhR
-null liver provides further support for the idea that C/EBPβ gene expression is RA responsive. This finding suggests that RA may regulate gene expression through modulation of C/EBPβ levels. It was also reported that expression of other members of the CCAAT/enhancer binding protein family such as C/EBPε are under RA control [38
], suggesting that this hormone regulates expression of this transcription factor family.
Schule et al. showed that addition of RARα reduced AP-1/DNA complex formation [39
], suggesting that RARα functions as a negative regulator of AP-1 responsive genes by interfering with their binding activity. More recently, Kim et al. observed that RAR and RXR associate with the serum response factor (SRF) in a ligand-dependent manner, and inhibit SRF transactivation activity [40
]. In addition, many studies have demonstrated that retinoid receptors inhibit the expression of several genes by interfering with the action of transcription factors [31
]. Specifically, RARs and RXRs have been shown to inhibit IL-6 gene expression by antagonizing the effect of NF-IL6, a CCAAT binding factor [41
]. The observed increase in RARα mRNA levels found in Ahr
-null mice liver may favor protein interaction between RARα and C/EBPβ, which may result in a decrease of C/EBPβ/CCAAT box complex formation. However no interaction between these two proteins was found.
Alternatively, other proteins may interact with C/EBPβ. It has been demonstrated that GADD153, a member of the CCAAT enhancer binding protein family, interacts and form complexes with C/EBPβ [34
]. On the other hand, GADD153 mRNA levels increase when NB4 and HL60 AML cell lines were treated with all-trans retinoic acid [44
]. In the Hepa-1 cells used in the present study, RA treatment increased GADD153 mRNA levels in a dose-dependent manner. Furthermore, we demonstrate that GADD153 binds C/EBPβ, serving as a dominant negative inhibitor, decreasing DNA-binding activity of C/EBPβ to the CCAAT box in the Raldh1
promoter. Interestingly, Fawcett et al. showed that, in rat pheochromocytoma PC12 cells, C/EBPβ acts as a positive regulator of GADD153 transcription, and provided evidence supporting an autoregulatory feedback loop in which expression of GADD153 suppresses its own transcription through its interaction with C/EBPβ [34
All together, these data suggest that Raldh1
transcription is under regulation of two linked negative feedback systems (). Raldh1, which plays a major role in RA synthesis, is under RARα and C/EBPβ transactivation control. Expression of both transcription factor genes is RA responsive, probably through RARα [23
] (). When RA levels increase, Raldh1 expression is inhibited. The high levels of this hormone increase C/EBPβ mRNA levels which increase GADD153 expression. GADD153 then interacts with C/EBPβ, decreasing DNA-binding activity of this transcription factor to the CCAAT box in the Raldh1
Fig. 10 Model of autoregulation of the Raldh1 gene promoter by heterodimerization of GADD153 and C/EBPβ. At low RA concentrations RARα and C/EBPβ transactivate the Raldh1 promoter (top panel). When RA levels increase, RARα transactivate (more ...)
RA levels observed in the AhR
-null mouse livers respond to a decrease on CYP2C39 transcription [19
]. Therefore, AhR
ligands would have the potential to alter the RA levels modifying not only Raldh1
gene expression, but also other genes under C/EBPβ-GADD145 autoregulatory feedback loop.
Finally, since C/EBPβ is a transcription factor that is critical to cellular differentiation, further studies will be necessary to elucidate the molecular mechanism by which RA induces C/EBPβ expression. The RARα RXRβ complex and C/EBPβ bind to specific sequences within the mouse Raldh1 gene promoter. These findings demonstrate that mouse Raldh1 is under similar transcriptional control as the human ALDH1 gene. We also showed that RA increases GADD153 and C/EBPβ mRNA levels, and promotes the interaction between these two proteins, resulting in the inhibition of Raldh1 expression.