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A growing amount of evidence suggests the involvement of ER (endoplasmic reticulum) stress in lipid metabolism and in the development of some liver diseases such as steatosis. The transcription factor SREBP-1 (sterol-regulatory-element-binding protein 1) modulates the expression of several enzymes involved in lipid synthesis. Previously, we showed that ER stress increased the SREBP-1a protein level in HepG2 cells, by inducing a cap-independent translation of SREBP-1a mRNA, through an IRES (internal ribosome entry site), located in its leader region. In the present paper, we report that the hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) interacts with 5'-UTR (untranslated region) of SREBP-1a mRNA, as an ITAF (IRES trans-acting factor), regulating SREBP-1a expression in HepG2 cells and in primary rat hepatocytes. Overexpression of hnRNP A1 in HepG2 cells and in rat hepatocytes increased both the SREBP-1a IRES activity and SREBP-1a protein level. Knockdown of hnRNP A1 by small interfering RNA reduced either the SREBP-1a IRES activity or SREBP-1a protein level. hnRNP A1 mediates the increase of SREBP-1a protein level and SREBP-1a IRES activity in Hep G2 cells and in rat hepatocytes upon tunicamycin- and thapsigargin-induced ER stress. The induced ER stress triggered the cytosolic relocation of hnRNP A1 and caused the increase in hnRNP A1 bound to the SREBP-1a 5'-UTR. These data indicate that hnRNP A1 participates in the IRES-dependent translation of SREBP-1a mRNA through RNA-protein interaction. A different content of hnRNP A1 was found in the nuclei from high-fat-diet-fed mice liver compared with standard-diet-fed mice liver, suggesting an involvement of ER stress-mediated hnRNP A1 subcellular redistribution on the onset of metabolic disorders.

SREBPs (sterol-regulatory-element-binding proteins) are a family of transcription factors that modulate the expression of several enzymes implicated in endogenous cholesterol, fatty acid, triacylglycerol and phospholipid synthesis. In the present study, evidence for SREBP-1 regulation at the translational level is reported. Using several experimental approaches, we have demonstrated that the 5'-UTR (untranslated region) of the SREBP-1a mRNA contains an IRES (internal ribosome entry site). Transfection experiments with the SREBP-1a 5'-UTR inserted in a dicistronic reporter vector showed a remarkable increase in the downstream cistron translation, through a cap-independent mechanism. Insertion of the SREBP-1c 5'-UTR in the same vector also stimulated the translation of the downstream cistron, but the observed effect can be ascribed, at least in part, to a cryptic promoter activity. Cellular stress conditions, such as serum starvation, caused an increase in the level of SREBP-1 precursor and mature form in both Hep G2 and HeLa cells, despite the overall reduction in protein synthesis, whereas mRNA levels for SREBP-1 were unaffected by serum starvation. Transfection experiments carried out with a dicistronic construct demonstrated that the cap-dependent translation was affected more than IRES-mediated translation by serum starvation. The thapsigargin- and tunicamycin-induced UPR (unfolded protein response) also increased SREBP-1 expression in Hep G2 cells, through the cap-independent translation mediated by IRES. Overall, these findings indicate that the presence of IRES in the SREBP-1a 5'-UTR allows translation to be maintained under conditions that are inhibitory to cap-dependent translation.

CiC (citrate carrier), a mitochondrial membrane protein, plays an important metabolic role by transporting acetyl-CoA into the cytosol for fatty acid and cholesterol synthesis. Several studies showed that CiC activity and expression is regulated by dietary fatty acids. In the present study we report data on the structural and functional characterization of the 5'-flanking region of the rat Cic gene. By transient transfection assays in H4IIE rat hepatoma cells, a PUFA (polyunsaturated fatty acids) response region has been identified within the CiC promoter. A cluster of putative binding sites for several transcription factors, composed of a NF-Y (nuclear factor-Y) site, an E-box-like site, a SRE1 (sterol regulatory element 1)-like site and four Sp1 (stimulatory protein 1) sites, was localized in the promoter region. Luciferase reporter gene and gel mobility shift assays indicated that a functional E-box-like, essential to the basal CiC promoter activity, confers responsiveness to activation by SREBP (SRE-binding protein)-1c. This study provides evidence for SREBP-1c as a principal target for PUFA regulation of CiC transcription. In H4IIE cells, overexpression of nSREBP (nuclear SREBP)-1c over-rides arachidonic acid (C(20:4, n-6)) suppression, but does not prevent the repression by docosahexaenoic acid (C(22:6, n-3)). ChIP (chromatin immunoprecipitation) assays in H4IIE cells showed that docosahexaenoic acid affects the binding of NF-Y, Sp1 and SREBP-1 to the PUFA response region of CiC promoter, whereas arachidonic acid alters only the binding of SREBP-1. Our data show that PUFA inhibition of hepatic Cic gene transcription is mediated not only by the nuclear level of SREBP-1c, but also might involve a reduction in Sp1 and NF-Y DNA binding, suggesting differential mechanisms in the Cic gene regulation by different PUFA.