NFE2L3 (NRF3): the Cinderella of the Cap'n'Collar transcription factors.

NFE2L3 [Nuclear factor (erythroid-derived 2)-like 3] or NRF3, a member of the Cap'n'Collar (CNC) family, is a basic-region leucine zipper (bZIP) transcription factor that was first identified over 10 years ago. Contrary to its extensively studied homolog NFE2L2 (NRF2), the regulation and function of the NFE2L3 protein have not yet attracted as much attention. Nevertheless, several recent reports have now shed light on the possible roles of NFE2L3. Structural and biochemical studies revealed a series of domains and modifications that are critical for its cellular regulation. The control of the subcellular localization of NFE2L3 appears to be essential for understanding its role in various cellular processes. Importantly, newer studies provide fascinating insights linking NFE2L3 to differentiation, inflammation, and carcinogenesis. Here, we present an overview of the current level of knowledge of NFE2L3 transcription factor biology in humans and mice. From being the Cinderella of the CNC transcription factors for many years, NFE2L3 may now rapidly come into its own.

A role for the peroxisomal 3-ketoacyl-CoA thiolase B enzyme in the control of PPARα-mediated upregulation of SREBP-2 target genes in the liver.

Peroxisomal 3-ketoacyl-CoA thiolase B (Thb) catalyzes the final step in the peroxisomal ß-oxidation of straight-chain acyl-CoAs and is under the transcription control of the nuclear hormone receptor PPARα. PPARα binds to and is activated by the synthetic compound Wy14,643 (Wy). Here, we show that the magnitude of Wy-mediated induction of peroxisomal ß-oxidation of radiolabeled (1-(14)C) palmitate was significantly reduced in mice deficient for Thb. In contrast, mitochondrial ß-oxidation was unaltered in Thb(-/-) mice. Given that Wy-treatment induced Acox1 and MFP-1/-2 activity at a similar level in both genotypes, we concluded that the thiolase step alone was responsible for the reduced peroxisomal ß-oxidation of fatty acids. Electron microscopic analysis and cytochemical localization of catalase indicated that peroxisome proliferation in the liver after Wy-treatment was normal in Thb(-/-) mice. Intriguingly, micro-array analysis revealed that mRNA levels of genes encoding cholesterol biosynthesis enzymes were upregulated by Wy in Wild-Type (WT) mice but not in Thb(-/-) mice, which was confirmed at the protein level for the selected genes. The non-induction of genes encoding cholesterol biosynthesis enzymes by Wy in Thb(-/-) mice appeared to be unrelated to defective SREBP-2 or PPARα signaling. No difference was observed in the plasma lathosterol/cholesterol ratio (a marker for de novo cholesterol biosynthesis) between Wy-treated WT and Thb(-/-) mice, suggesting functional compensation. Overall, we conclude that ThA and SCPx/SCP2 thiolases cannot fully compensate for the absence of ThB. In addition, our data indicate that ThB is involved in the regulation of genes encoding cholesterol biosynthesis enzymes in the liver, suggesting that the peroxisome could be a promising candidate for the correction of cholesterol imbalance in dyslipidemia.

Nfe2l3 (Nrf3) deficiency predisposes mice to T-cell lymphoblastic lymphoma.

We have previously generated mice deficient for Nfe213 (NF-E2 p45 related factor 3 or Nrf3), a member of the cap 'n' collar family of basic-leucine zipper transcription factors. To examine whether Nrf3 is involved in chemical-induced carcinogenesis, we exposed the mice to benzo[a]pyrene (B[a]P), a carcinogen found in cigarette smoke. Contrary to wild-type mice, Nrf3-null animals are highly susceptible to B[a]P, exhibiting significantly increased mortality. Pathology analysis of affected tissue sections revealed a high incidence of T-cell lymphoblastic lymphoma in B[a]P-treated Nrf3(-/-) mice. Lymphoblastic lymphoma occasionally metastasized into the lung as demonstrated by perivascular malignant lymphocytic infiltration. Together, our studies show that the absence of Nrf3 predisposes mice to lymphoma development, suggesting a protective role of this transcription factor in hematopoietic malignancies. Our data demonstrate the first in vivo function of Nrf3 and its link to tumor development. Nrf3-deficient mice may serve as a preclinical mouse model to study carcinogen-induced lymphomagenesis.

Nrf3-deficient mice are not protected against acute lung and adipose tissue damages induced by butylated hydroxytoluene.

We found that both wild type and Nrf3 (NF-E2-related factor 3) deficient mice are sensitive to BHT single administration exhibiting respiratory distress and considerably lose body weight following treatment. At time of sacrifice, the BHT-treated Nrf3-/- mice had lost significantly more body weight than their WT counterparts. In the lung, transcript levels of the transcription factors Nrf1, Nrf2 and Nrf3 were differentially regulated by BHT treatment. In addition, genes implicated in adipogenesis were repressed following BHT exposure in the white adipose tissue. Together, our data provide the first evidence that BHT exposure not only affects lung function but also leads to impaired adipogenesis in adipocytes.

Modulation of the hepatic fatty acid pool in peroxisomal 3-ketoacyl-CoA thiolase B-null mice exposed to the selective PPARalpha agonist Wy14,643.

The peroxisomal 3-ketoacyl-CoA thiolase B (Thb) gene was previously identified as a direct target gene of PPARalpha, a nuclear hormone receptor activated by hypolipidemic fibrate drugs. To better understand the role of ThB in hepatic lipid metabolism in mice, Sv129 wild-type and Thb null mice were fed or not the selective PPARalpha agonist Wy14,643 (Wy). Here, it is shown that in contrast to some other mouse models deficient for peroxisomal enzymes, the hepatic PPARalpha signaling cascade in Thb null mice was normal under regular conditions. It is of interest that the hypotriglyceridemic action of Wy was reduced in Thb null mice underlining the conclusion that neither thiolase A nor SCPx/SCP2 thiolase can fully substitute for ThB in vivo. Moreover, a significant increased in the expression of lipogenic genes such as Stearoyl CoA Desaturase-1 (SCD1) was observed in Thb null mice fed Wy. Elevation of Scd1 mRNA and protein levels led to higher SCD1 activity, through a molecular mechanism that is probably SREBP1 independent. In agreement with higher SCD1, enrichment of liver mono-unsaturated fatty acids of the n-7 and n-9 series was found in Thb null mice fed Wy. Overall, we show that the reduced peroxisomal beta-oxidation of fat observed in Thb null mice fed Wy is associated with enhanced hepatic lipogenesis, through the combined elevation of microsomal SCD1 protein and activity. Ultimately, not only the amount but also the quality of the hepatic fatty acid pool is modulated upon the deletion of Thb.

Identification of interleukin-1beta regulated genes in uterine smooth muscle cells.

We analyzed the response of uterine smooth muscle cells to interleukin-1beta (IL-1beta). We first showed that PHM1-31 myometrial cells, our cellular model, are contractile. To determine the molecular mechanisms of uterine smooth muscle cell activation by proinflammatory cytokines, we performed genechip expression array profiling studies of PHM1-31 cells in the absence and the presence of IL-1beta. In total, we identified 198 known genes whose mRNA levels are significantly modulated (> 2.0-fold change) following IL-1beta exposure. We confirmed the expression changes for selected genes by independent mRNA and protein analysis. The group of genes induced by IL-1beta includes transcription factors and inflammatory response genes such as nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFkappaB), pentraxin-related gene (PTX3), and tumor necrosis factor alpha-induced protein 3/A20 (TNFAIP3/A20). We also found up-regulation of chemokines like C-X-C motif ligand 3 (CXCL3) and extracellular matrix remodeling signaling molecules like tenascin C (TNC). Our data suggest that IL-1beta elicits the rapid activation of a cellular network of genes particularly implicated in inflammatory response that may create a cellular environment favorable for myometrial cell contraction. Our results provide novel insights into the mechanisms of uterine smooth muscle cell regulation and possibly infection-induced preterm labor.

Endoplasmic reticulum association and N-linked glycosylation of the human Nrf3 transcription factor.

We have analysed the molecular and cellular regulation of the basic-leucine zipper (bZIP) transcription factor Nrf3 (NFE2-Related Factor 3). Cycloheximide studies revealed a rapid turnover of Nrf3. We showed that the proteasome inhibitor MG-132 increases Nrf3 protein levels. Furthermore, we demonstrated that Nrf3 is an N-glycosylated protein associated with the endoplasmic reticulum. Thus, our studies provide the first evidence of a post-translational modification of Nrf3.

Targeted disruption of the peroxisomal thiolase B gene in mouse: a new model to study disorders related to peroxisomal lipid metabolism.

The peroxisomal beta-oxidation system consists of four steps catalysed by three enzymes: acyl-CoA oxidase, 3-hydroxyacyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (multifunctional enzyme) and thiolase. In humans, thiolase activity is encoded by one gene, whereas in rodents, three enzymes encoded by three distinct genes (i.e. thiolase A, thiolase B and SCP2/thiolase) catalyse the thiolase activity. So far, acyl-CoA oxidase- and multifunctional enzyme-deficient patients have been identified and knock-out mice for these genes have been produced. Conversely, no isolated thiolase-deficient patient has been found, and no thiolase (A or B)-deficient mice have been generated. Hence, to better understand the cause of isolated human thiolase deficiency, we disrupted the catalytic site of the mouse thiolase B by homologous recombination in order to analyse the phenotype of these thiolase B-deficient mice. Mice, made homozygous for the mutation, lack expression of thiolase B mRNA and are viable, fertile and healthy at birth. They exhibit no detectable phenotype defects and no compensation, rather a slight decrease in other peroxisomal thiolase (thiolase A and SCPx) mRNAs, was found.

Effects of peroxisome proliferator-activated receptor alpha activation on pathways contributing to cholesterol homeostasis in rat hepatocytes.

Peroxisome proliferator-activated receptor alpha (PPARalpha) activation by fibrates controls expression of several genes involved in hepatic cholesterol metabolism. Other genes could be indirectly controlled in response to changes in cellular cholesterol availability. To further understand how fibrates may affect cholesterol synthesis, we investigated in parallel the changes in the metabolic pathways contributing to cholesterol homeostasis in liver. Ciprofibrate increased HMG-CoA reductase and FPP synthase mRNA levels in rat hepatocytes, together with cholesterogenesis from [(14)C] acetate and [(3)H] mevalonate. The up-regulation observed in fenofibrate- and WY-14,643-treated mice was abolished in PPARalpha-null mice, showing an essential role of PPARalpha. Among the three sterol regulatory element-binding protein (SREBP) mRNA species, only SREBP-1c level was significantly increased. In ciprofibrate-treated hepatocytes, cholesterol efflux was decreased, in parallel with cholesteryl ester storage and bile acids synthesis. As expected, AOX expression was strongly induced, supporting evidence of the peroxisome proliferation. Taken together, these results show that fibrates can cause cholesterol depletion in hepatocytes, possibly in part as a consequence of an important requirement of cholesterol for peroxisome proliferation, and increase cholesterogenesis by a compensatory phenomenon afterwards. Such cholesterogenesis regulation could occur in vivo, in species responsive to the peroxisome proliferative effect of PPARalpha ligands.

Molecular cloning, gene structure and expression profile of two mouse peroxisomal 3-ketoacyl-CoA thiolase genes.

BACKGROUND: In rats, two peroxisomal 3-ketoacyl-CoA thiolase genes (A and B) have been cloned, whereas only one thiolase gene is found in humans. The aim of this study was thus to clone the different mouse thiolase genes in order to study both their tissue expression and their associated enzymatic activity. RESULTS: In this study, we cloned and characterized two mouse peroxisomal 3-ketoacyl-CoA thiolase genes (termed thiolase A and B). Both thiolase A and B genes contain 12 exons and 11 introns. Using RNA extracted from mouse liver, we cloned the two corresponding cDNAs. Thiolase A and B cDNAs possess an open reading frame of 1272 nucleotides encoding a protein of 424 amino acids. In the coding sequence, the two thiolase genes exhibited approximately equal to 97% nucleotide sequence identity and approximately equal to 96% identity at the amino acid level. The tissue-specific expression of the two peroxisomal 3-ketoacyl-CoA thiolase genes was studied in mice. Thiolase A mRNA was mainly expressed in liver and intestine, while thiolase B mRNA essentially exhibited hepatic expression and weaker levels in kidney, intestine and white adipose tissue. Thiolase A and B expressions in the other tissues such as brain or muscle were very low though these tissues were chiefly involved in peroxisomal disorders. At the enzymatic level, thiolase activity was detected in liver, kidney, intestine and white adipose tissue but no significant difference was observed between these four tissues. Moreover, thiolase A and B genes were differently induced in liver of mice treated with fenofibrate. CONCLUSION: Two mouse thiolase genes and cDNAs were cloned. Their corresponding transcripts are mostly expressed in the liver of mice and are differently induced by fenofibrate.