is a key enzyme of hepatic lipogenesis responsible for the synthesis of long-chain saturated fatty acids. This enzyme is mainly regulated at the transcriptional level by nutrients and hormones. In particular, glucose, insulin, and T 3 increase FAS activity, whereas glucagon and saturated and polyunsaturated fatty acids decrease it. In the present study we show that, in liver, T 3 and insulin were able to activate FAS enzymatic activity, mRNA expression, and gene transcription. We localized the T 3 response element (TRE) that mediates the T3 genomic effect, on the FAS promoter between Ϫ741 and Ϫ696 bp that mediates the T 3 genomic effect. We show that both T3 and insulin regulate FAS transcription via this sequence. The TRE binds a TR/RXR heterodimer even in the absence of hormone, and this binding is increased in response to T 3 and/or insulin treatment. The use of H7, a serine/threonine kinase inhibitor, reveals that a phosphorylation mechanism is implicated in the transcriptional regulation of FAS in response to both hormones. Specifically, we show that T 3 is able to modulate FAS transcription via a nongenomic action targeting the TRE through the activation of a PI 3-kinase-ERK1/2-MAPK-dependent pathway. Insulin also targets the TRE sequence, probably via the activation of two parallel pathways: Ras/ERK1/2 MAPK and PI 3-kinase/Akt. Finally, our data suggest that the nongenomic actions of T 3 and insulin are probably common to several TREs, as we observed similar effects on a classical DR4 consensus sequence. fatty acid synthase; triiodothyronine; insulin; triiodothyronine response element; phosphoinositide 3-kinase; extracellular signal-regulated kinase-1/2 mitogen-activated protein kinase LIPOGENESIS CONVERTS DIETARY CARBOHYDRATES to fatty acids primarily in liver (28). Insulin and triiodothyronine (T 3 ) are involved in mediating the effects of diet on lipogenesis in vivo (34). Hepatic lipogenesis is increased in hyperthyroid states or in response to T 3 injection (10,15,19,24,25,28,62,71,76,83) as well as in hyperinsulinemic subjects (80). In vivo, these two hormones are also involved in the long-term regulation of lipogenic enzymes activities such as fatty acid synthase (37).Fatty acid synthase (FAS; EC.2.3.1.85) is a key enzyme in hepatic lipogenesis. In the presence of NADPH, this multifunctional enzyme catalyzes the conversion of acetyl-CoA and malonyl-CoA into long-chain saturated fatty acids such as palmitate and stearate (92). The de novo synthesis of fatty acids in human and chicken takes place mainly in the liver (30, 58), whereas in rodents the adipose tissue is also lipogenic (30). In vertebrates, FAS is a homodimer made of two identical peptide chains of ϳ260 kDa (85, 91), located in the cytoplasm of the cell (31). FAS is encoded by a unique gene that generates only one mRNA in mouse (73) and two in chicken and rat, as a result of alternative splicing (3). In the liver, the activity of FAS, like most lipogenic enzymes (95), is regulated through nutrients and hormones. Starvation causes a decrea...

BackgroundCystinosis is a rare disorder caused by recessive mutations of the CTNS gene. Current therapy decreases cystine accumulation, thus slowing organ deterioration without reversing renal Fanconi syndrome or preventing eventual need for a kidney transplant.15-20% of cystinosis patients harbour at least one nonsense mutation in CTNS, leading to premature end of translation of the transcript. Aminoglycosides have been shown to permit translational read-through but have high toxicity level, especially in the kidney and inner ear. ELX-02, a modified aminoglycoside, retains it read-through ability without the toxicity.Methods and findingsWe ascertained the toxicity of ELX-02 in cells and in mice as well as the effect of ELX-02 on translational read-through of nonsense mutations in cystinotic mice and human cells. ELX-02 was not toxic in vitro or in vivo, and permitted read-through of nonsense mutations in cystinotic mice and human cells.ConclusionsELX-02 has translational read-through activity and produces a functional CTNS protein, as evidenced by reduced cystine accumulation. This reduction is comparable to cysteamine treatment. ELX-02 accumulates in the kidney but neither cytotoxicity nor nephrotoxicity was observed.

Background: Hereditary Wilms tumors are preceded by WT1(Ϫ) clones with an inhibitory chromatin histone pattern established by EZH2. Results: In amniotic mesenchymal stem cells, WT1 suppresses EZH2, derepresses ␤-catenin (CTNNB1), and enhances responsiveness to WNT9b. Conclusion: WT1 regulates transition from the epigenetically silenced chromatin state. Significance: Developmental blockade in nephrogenic rests may be mediated by loss of the WT1-EZH2-CTNNB1 axis.

Fatty acid synthase (FAS) is responsible for the de novo synthesis of palmitate and stearate. This enzyme is activated by insulin and T(3), and inhibited by fatty acids. In this study, we show that insulin and T(3) have an inducing effect on FAS enzymatic activity, which is synergetic when both hormones are present. Octanoate and hexanoate specifically inhibit this hormonal effect. A similar inhibitory effect is observed at the level of protein expression. Transient transfections in HepG2 cells revealed that hexanoate inhibits, at least in part, FAS at a transcriptional level targeting the T(3) response element (TRE) on the FAS promoter. The effect of C6 on FAS expression cannot be attributed to a modification of insulin receptor activation or to a decrease in T(3) entry in the cells. Using bromo-hexanoate, we determined that hexanoate needs to undergo a transformation in order to have an effect. When incubating cells with triglyceride-hexanoate or carnitine-hexanoate, no effect on the enzymatic activity induced by insulin and T(3) is observed. A similar result was obtained when cells were incubated with betulinic acid, an inhibitor of the diacylglycerol acyltransferase. However, the incubation of cells with Triacsin C, a general inhibitor of acyl-CoA synthetases, completely reversed the inhibitory effect of hexanoate. Our results suggest that in hepatic cells, hexanoate needs to be activated into a CoA derivative in order to inhibit the insulin and T(3)-induced FAS expression. This effect is partially transcriptional, targeting the TRE on the FAS promoter.

Hereditary forms of Wilms arise from developmentally arrested clones of renal progenitor cells with biallelic mutations of WT1; recently, it has been found that Wilms tumors may also be associated with biallelic mutations in DICER1 or DROSHA, crucial for miRNA biogenesis. We have previously shown that a critical role for WT1 during normal nephrogenesis is to suppress transcription of the Polycomb group protein, EZH2, thereby de-repressing genes in the differentiation cascade. Here we show that WT1 also suppresses translation of EZH2. All major WT1 isoforms induce an array of miRNAs, which target the 3 UTR of EZH2 and other Polycomb-associated transcripts. We show that the WT1(؉KTS) isoform binds to the 5 UTR of EZH2 and interacts directly with the miRNA-containing RISC to enhance post-transcriptional inhibition. These observations suggest a novel mechanism through which WT1 regulates the transition from resting stem cell to activated progenitor cell during nephrogenesis. Our findings also offer a plausible explanation for the fact that Wilms tumors can arise either from loss of WT1 or loss of miRNA processing enzymes.In 1879, William Osler reported two pediatric patients from Montreal with massive kidney tumors containing bands of muscle-like tissue mixed with epithelial elements (35). Twenty years later, Max Wilms published his celebrated monograph recognizing the malignancy as a unique "mischgeschwulste der Niere" (mixed tumor of the kidney), composed of mixed stromal, epithelial, and undifferentiated mesenchymal cells. This "triphasic" histology suggests that Wilms tumors arise from developmentally arrested stem cells of the metanephric mesenchyme that occasionally exhibit abortive differentiation toward a stromal or an epithelial cell fate. It follows that a disturbance of molecular events governing the transition from renal progenitor cells into these differentiated lineages must be central to the pathogenesis of Wilms tumor. Wilms tumor (WT)2 is the most common form of pediatric kidney cancer and affects about 1:10,000 children in North America (1). In a subset of these patients, a germline deletion of the transcription factor, WT1, is accompanied by a somatic mutation of the second WT1 allele, giving rise to clones of incompetent stem cells adjacent to the malignant tumor (2). These "nephrogenic rests" are thought to represent embryonic renal progenitors that have failed to respond to inductive WNT signals during embryogenesis. The pre-malignant cell clusters may persist within the normal kidney (3) until a constitutively activating mutation of the ␤-catenin gene (CTNNB1) (17, 18) bypasses the need for a normal WNT signal and drives un-regulated rapid cell growth (4). Wilms tumors retain a chromatin pattern resembling embryonic stem cells in which genes of the differentiation cascade are broadly silenced by tri-methylated lysine 27 residues in the H3 histone associated with their promoter regions (5). We recently showed that the WT1 isoforms lacking the three amino acid insertion lysine-threonine-serine b...