Upregulation of gene expression

Nuclear receptors (NRs) are members of a superfamily of transcription factors that share a common domain organization and sequence similitude. To date, over 300 NRs have been cloned. Most of them do not have an identifiable relevant ligand (orphan receptors). Others are ligand-inducible and respond to endogenous or exogenous lipophilic molecules. These factors are able to bind specific response elements in the vicinity of the genes on which they act. The presence of bound ligands induces a transconformation state that makes them recruit co-activators in place of co-repressors that they bind in the absence of ligand. Cross-regulations between different members of this family are due to their ability to dimerize and the fact that they share similar affinity for a small number of co-activators and co-repressors. Here, two of them will be more particularly quoted, PPARs and HNF4, since they are directly linked to the fatty acid regulation of gene expression. However, other nuclear receptors such as thyroid hormone receptors or chicken ovalbumin upstream promoter transcription factors have been consistently shown to be able to bind FAs (Duplus et al., 2000; Duplus and Forest, 2002).

Peroxisome proliferator-activated receptors are members of the superfamily of nuclear receptors, which are ligand-activated transcription factors. In mammals, three subtypes have been described. Their tissue distribution largely differs: PPAR beta is expressed in most cells, while the expression of other subtypes is more restricted (mainly liver, heart and muscle for PPAR alpha and adipokines for PPAR gamma). Rat PPAR alpha cDNA was first cloned from the liver and is the only true peroxisome proliferator. Human PPAR alpha and all subtypes have been named because of high sequence similitude. All of them are transcriptionaly active only after heterodimerization with another nuclear receptor, the 9-cis retinoic acid activated retinoid X receptor (RXR). RXR acts as a heterodimerization partner with other nuclear receptors, some of them able to bind lipid derivatives such as thyroid hormone receptors (TR) or LXR, which hints a possible interplay between nuclear receptors with regard to ligand concentrations. In addition, PPARs generally show low ligand specificity, being activated by several long chain saturated or unsaturated fatty acids and by eicosanoids. This is certainly due to a generous pocket that also allows binding to many other endogenous or pharmacological compounds (fibrate or thiazolidinedione).

The ubiquitous expression of PPAR beta made the identification of its function elusive. However, it has recently been shown that a PPAR beta-specific ligand promotes lipid accumulation in human macrophages, which can be considered potentially pro-atherogenic (Vosper et al., 2001). In contrast, the high and almost selective expression of PPAR gamma in adipocytes shows that it is likely to play a major role in the differentiation of these cells (Tontonoz et al., 1994; Grimaldi, 2001; Kliewer et al., 2001; Ferre, 2004). But there is a concern about its role in energy metabolism in these cells (Walczak and Tontonoz, 2002). Some genes (for example, those coding for the glucose transporter GLUT4, the acyl CoA synthase or the phospho-enol-pyruvate-CK) are involved in the activation and esterification of fatty acids and display specific DNA elements able to bind PPAR. However, simply because FA are potential ligands of PPARs does not necessarily imply that transcriptional effects of FA are mediated through these nuclear receptors. This point has been clearly illustrated in the PEP-CK gene. For example, several effects induced by FA are PPAR-independent and are probably modulated by other transcription factors (Duplus and Forest, 2002).

The putative role of PPARs as FA-induced nuclear receptors in several cells is more directly supported by studies on PPAR alpha. Indeed, it has been possible to delete this gene in mice and these animals subsequently lost the ability to increase their fatty acids oxidation when treated with fibrate, a PPAR alpha ligand. In addition, although the phenotype of normally fed null mice was not fundamentally different from wild type, starvation induced major differences. Null mice presented an impaired utilization of fatty acids that led to hepatic and cardiac steatosis. These observations are sustained by mechanistic studies on genes that are obviously involved in FA transport, synthesis of CoA derivatives and the subsequent peroxisomal degradation of very long chain fatty acids (the example of the liver acyl CoA oxidase is particularly clear). The expressions of genes coding for some key enzymes involved in mainstream mitochondrial oxidation and synthesis of ketone bodies are also activated by PPAR alpha ligand. The key regulatory enzyme of mitochondrial oxidation is carnitine palmitoyl transferase type I, which allows the activated FA to enter the mitochondria. In primary hepatocytes, the expression of this gene is clearly induced by fibrates and long chain fatty acids, whether saturated or not. Nevertheless, the careful deciphering of regulatory sequences in the vicinity of the CPT 1 gene demonstrated that FA and fibrates act through different elements, suggesting that the FA-induced overexpression of CPT 1 is PPAR-independent process. This study provides us with another example of the difficulty of unequivocally ascribing the FA-induced regulation of a given gene to PPARs, even when pharmacological effects of PPAR-specific ligands seem to permit such a deduction.

Hepatic Nuclear Factor 4 (HNF-4) has long been considered an orphan receptor until structural studies reported it was able to constitutively bind FAs. Recent studies reported that the transcriptional activity of this receptor could be modulated by long chain FA when a reporter gene was fused with response elements issued from the human ApoC-III promoter (Pastier et al., 2002). This activation was paralleled by the binding of cognate acyl-CoA thioesters to the ligand domain of HNF-4. This process was saturation dependent since palmitoyl-CoA derivative greatly enhanced the transcriptional activity as well as the binding of HNF4 to its specific DNA site, while PUFA and their respective acyl-CoA derivatives displayed a clear inhibition of transcriptional activity, together with a diminished DNA binding. It has also been recently demonstrated that the Apo A-IV gene transcription in enterocyte cell cultures is dependent on the apical supply of complexes of micelles mimicking the composition of duodenal micelles. This increase was clearly abolished when a negative dominant form of HNF-4 was transfected, which strongly suggests the involvement of the nuclear receptor in this regulation (Carriere et al., 2005).

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