HDAC1-Dependent Repression of Markers of Hepatocytes and P21 Is Involved in Development of Pediatric Liver Cancer.

BACKGROUND & AIMS: Epigenetic regulation of gene expression plays a critical role in the development of liver cancer; however, the molecular mechanisms of epigenetic-driven liver cancers are not well understood. The aims of this study were to examine molecular mechanisms that cause the dedifferentiation of hepatocytes into cancer cells in aggressive hepatoblastoma and test if the inhibition of these mechanisms inhibits tumor growth. METHODS: We have analyzed CCAAT/Enhancer Binding Protein alpha (C/EBPα), Transcription factor Sp5, and histone deacetylase (HDAC)1 pathways from a large biobank of fresh hepatoblastoma (HBL) samples using high-pressure liquid chromatography-based examination of protein-protein complexes and have examined chromatin remodeling on the promoters of markers of hepatocytes and p21. The HDAC1 activity was inhibited in patient-derived xenograft models of HBL and in cultured hepatoblastoma cells and expression of HDAC1-dependent markers of hepatocytes was examined. RESULTS: Analyses of a biobank showed that a significant portion of HBL patients have increased levels of an oncogenic de-phosphorylated-S190-C/EBPα, Sp5, and HDAC1 compared with amounts of these proteins in adjacent regions. We found that the oncogenic de-phosphorylated-S190-C/EBPα is created in aggressive HBL by protein phosphatase 2A, which is increased within the nucleus and dephosphorylates C/EBPα at Ser190. C/EBPα-HDAC1 and Sp5-HDAC1 complexes are abundant in hepatocytes, which dedifferentiate into cancer cells. Studies in HBL cells have shown that C/EBPα-HDAC1 and Sp5-HDAC1 complexes reduce markers of hepatocytes and p21 via repression of their promoters. Pharmacologic inhibition of C/EBPα-HDAC1 and Sp5-HDAC1 complexes by Suberoylanilide hydroxamic acid (SAHA) and small interfering RNA-mediated inhibition of HDAC1 increase expression of hepatocyte markers, p21, and inhibit proliferation of cancer cells. CONCLUSIONS: HDAC1-mediated repression of markers of hepatocytes is an essential step for the development of HBL, providing background for generation of therapies for aggressive HBL by targeting HDAC1 activities.

CCAAT/enhancer-binding protein alpha (CEBPA) gene haploinsufficiency does not alter hematopoiesis or induce leukemia in Lck-CALM/AF10 transgenic mice.

Although rare, CALM/AF10 is a chromosomal rearrangement found in immature T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia, and mixed phenotype acute leukemia of T/myeloid lineages with poor prognosis. Moreover, this translocation is detected in 50% of T-ALL patients with gamma/delta T cell receptor rearrangement, frequently associated with low expression of transcription factor CCAAT/enhancer-binding protein alpha (CEBPA). However, the relevance of CEBPA low expression for CALM/AF10 leukemogenesis has not yet been evaluated. We generated double mutant mice, which express the Lck-CALM/AF10 fusion gene and are haploinsufficient for the Cebpa gene. To characterize the hematopoiesis, we quantified hematopoietic stem cells, myeloid progenitor cells, megakaryocyte-erythrocyte progenitor cells, common myeloid progenitor cells, and granulocyte-macrophage progenitor cells. No significant difference was detected in any of the progenitor subsets. Finally, we tested if Cebpa haploinsufficiency would lead to the expansion of Mac-1+/B220+/c-Kit+ cells proposed as the CALM/AF10 leukemic progenitor. Less than 1% of bone marrow cells expressed Mac-1, B220, and c-Kit with no significant difference between groups. Our results showed that the reduction of Cebpa gene expression in Lck-CALM/AF10 mice did not affect their hematopoiesis or induce leukemia. Our data corroborated previous studies suggesting that the CALM/AF10 leukemia-initiating cells are early progenitors with lymphoid/myeloid differentiating potential.

α, ß-Amyrin, a pentacyclic triterpenoid from Protium heptaphyllum suppresses adipocyte differentiation accompanied by down regulation of PPARγ and C/EBPα in 3T3-L1 cells.

Previous studies have reported the anti-obesity effects of α, ß-Amyrin in high fat-fed mice. This study aimed to evaluate whether α, ß-Amyrin has an anti-adipogenic effect in 3T3-L1 murine adipocytes and to explore the possible underlying mechanisms. 3T3-L1 pre-adipocytes were differentiated in a medium containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthine. Cytotoxicity of α, ß-Amyrin was assessed by MTT assay. Lipid content in adipocytes was determined by Oil-Red O staining. In addition, the protein expression levels of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding proteins alpha (C/EBPα), beta (C/EBPß), and delta (C/EBPδ) and glucose transporter 4 (GLUT4) were determined by qRT-PCR and western blot analysis. Oil-Red O staining revealed markedly reduced fat accumulation by α, ß-Amyrin (6.25-50 µg/mL) without affecting cell viability. Furthermore, our results indicate that α, ß-Amyrin can significantly suppress the adipocyte differentiation by downregulating the expression levels of adipogenesis-related key transcription factors such as PPARγ and C/EBPα, but not C/EBPß or C/EPBδ. In addition, the protein expression of membrane GLUT4 in 3T3- L1 adipocytes treated with α, ß-Amyrin was significantly higher than in control cells, indicating that α, ß-Amyrin augments glucose uptake. These findings suggest that α, ß-Amyrin exerts an anti-adipogenic effect principally via modulation of lipid and carbohydrate metabolism in 3T3-L1cells. The present in vitro findings, taken together with our earlier observation of the anti-obesity effect in vivo, suggest that α, ß-Amyrin can be developed as a new therapeutic agent for treatment and prevention of obesity.

CCAAT/enhancer-binding protein alpha (CEBPA) gene haploinsufficiency does not alter hematopoiesis or induce leukemia in Lck-CALM/AF10 transgenic mice

Although rare, CALM/AF10 is a chromosomal rearrangement found in immature T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia, and mixed phenotype acute leukemia of T/myeloid lineages with poor prognosis. Moreover, this translocation is detected in 50% of T-ALL patients with gamma/delta T cell receptor rearrangement, frequently associated with low expression of transcription factor CCAAT/enhancer-binding protein alpha (CEBPA). However, the relevance of CEBPA low expression for CALM/AF10 leukemogenesis has not yet been evaluated. We generated double mutant mice, which express the Lck-CALM/AF10 fusion gene and are haploinsufficient for the Cebpa gene. To characterize the hematopoiesis, we quantified hematopoietic stem cells, myeloid progenitor cells, megakaryocyte-erythrocyte progenitor cells, common myeloid progenitor cells, and granulocyte-macrophage progenitor cells. No significant difference was detected in any of the progenitor subsets. Finally, we tested if Cebpa haploinsufficiency would lead to the expansion of Mac-1+/B220+/c-Kit+ cells proposed as the CALM/AF10 leukemic progenitor. Less than 1% of bone marrow cells expressed Mac-1, B220, and c-Kit with no significant difference between groups. Our results showed that the reduction of Cebpa gene expression in Lck-CALM/AF10 mice did not affect their hematopoiesis or induce leukemia. Our data corroborated previous studies suggesting that the CALM/AF10 leukemia-initiating cells are early progenitors with lymphoid/myeloid differentiating potential.

Immunometabolic regulation by triterpenes of Eucalyptus tereticornis in adipose tissue cell line models.

BACKGROUND: Eucalyptus tereticornis Sm (Myrtaceae) is a plant used in traditional medicine to control obesity, insulin resistance and diabetes. Chronic adipose tissue inflammation is involved in generating insulin resistance, the greatest risk factor in developing type 2 diabetes mellitus and cardiovascular disease. In the present study, a mixture of triterpenes, as obtained from the starting plant material, was evaluated in inflamed adipose tissue cells models. AIM: Our goal is to advance into the understanding, at the cellular level, of the immunometabolic effects of the triterpene mixes from Eucalyptus tereticornis in in vitro models of mouse and human adipose tissues. METHODS: Triterpene mixes were obtained from Eucalyptus tereticornis leaves by organic extraction. The major compounds of these mixes were identified by 1H NMR and 13C NMR in addition to HPLC using primary and secondary standards of ursolic acid, oleanolic acid and ursolic acid lactone. To provide an approach for evaluating the cellular and molecular mechanisms through which triterpene mixes act to modify the metabolic processes associated with obesity, mouse macrophage and adipocyte cell lines, human macrophage cell line and primary culture of human adipocytes were used as models. RESULTS: Adipocytes treated with the two natural chemically characterized triterpene mixes partially reduce lipogenesis and leptin expression. Additionally, an increase in the transcriptional expression of PPARγ, and C/EBPα is observed. In macrophages, these triterpene mixes, decrease the transcriptional and translational expression of pro-inflammatory cytokines, such as interleukin-6 (IL-6), interleukin 1ß (IL-1ß) and tumoral necrosis factor α (TNFα). Conditioned medium of 3T3-L1 adipocytes treated with the triterpene mix shows a stronger anti-inflammatory response on activated J774A.1 macrophages. CONCLUSION: The mixtures of the three triterpenes in the proportions obtained from the plant material may act on different components of the cell, generating a different response, which, in some cases, is more powerful than that seen when exposure to only two triterpenes. It makes this three triterpenes mix a good phytotherapeutic prototype for pathologies as complex as those associated with obesity.

Obesity modifies bone marrow microenvironment and directs bone marrow mesenchymal cells to adipogenesis.

OBJECTIVE: To investigate the role of obesity on the bone marrow microenvironment and evaluate its possible impact on the adipogenic potential of mesenchymal stem cells (MSC). METHODS: C57BL/6 male mice were fed with a high-fat diet (HFD) for 10 weeks. Femurs and tibiae were collected, and bone marrow mesenchymal stem cells (BM-MSC) were isolated and analyzed for proliferative potential, immunophenotype, and expression of adipogenesis markers. Their capacity to produce extracellular matrix proteins and proinflammatory cytokines in vitro was also evaluated. RESULTS: HFD mice presented a significant increase in bone marrow cellularity and higher tumor necrosis factor-α production in vitro. BM-MSC from HFD mice had higher proliferative capacity, produced more extracellular matrix proteins associated with adipogenesis, collagen I, and collagen IV, and showed increased constitutive expression of adipogenic markers, peroxisome proliferator-activated receptor-γ, and CCAAT/enhanced binding protein family-α, without changes in preadipocyte factor-1 expression. Incubation with adipocyte-differentiation medium induced further increase in CCAAT/enhanced binding protein family-α and augmented adiponectin expression in obese BM-MSC. These alterations did not result in increased adipogenic differentiation within the bone marrow. Moreover, BM-HSC from HFD mice, co-cultivated with BM-MSCs from lean mice, exerted paracrine effects on these cells, inducing augment of peroxisome proliferator-activated receptor-γ. CONCLUSIONS: The data suggest that obesity promotes an inflammatory microenvironment in bone marrow that commits BM-MSC to adipogenesis.

AMFR gene silencing inhibits the differentiation of porcine preadipocytes.

Our study clarifies the role of the autocrine motility factor receptor (AMFR) gene in porcine preadipocyte differentiation. AMFR-siRNA was transfected into porcine preadipocytes and the preadipocytes were induced to differentiation. Subsequently, qRT-PCR was conducted to examine changes in mRNA expression of a series of genes in porcine preadipocytes, including AMFR, sterol-regulatory element-binding protein-1a (SREBP1a), SREBP2, insulin-induced gene 1 (Insig1), and Insig2. Expression changes in the mRNA of genes regulating adipocyte differentiation were also analyzed using qRT-PCR, including peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), and Kruppel-like factor 2 (KLF2). Western blot analysis was conducted to examine the changes in AMFR protein expression in porcine preadipocytes. Additionally, morphological changes in differentiated porcine preadipocytes were examined by oil red O staining, and changes in optical density (OD) values were measured using an ultraviolet spectrophotometer. At 24 h after transfection with AMFR-siRNA, AMFR mRNA expression significantly reduced (P < 0.01), and AMFR protein expression markedly decreased (P < 0.05). The mRNA expression of SREBP1a, SREBP2, Insig1, and C/EBPα was significantly reduced (P < 0.01), whereas the expression of KLF2 mRNA was significantly elevated (P < 0.01). After induction of preadipocyte differentiation, the number of lipid droplets decreased in the AMFR-silenced group, and the OD value markedly reduced (P < 0.05). In addition, the expression of C/EBPα mRNA significantly decreased (P < 0.05), whereas the expression of KLF2 mRNA considerably increased (P < 0.05). Taken together, silencing of the AMFR gene inhibits the differentiation of porcine preadipocytes.

Tangeritin inhibits adipogenesis by down-regulating C/EBPα, C/EBPß, and PPARγ expression in 3T3-L1 fat cells.

The treatment of obese patients is a topic investigated by an increasing number of researchers. This study aimed to elucidate the possible inhibitory effect of tangeritin on the development and function of fat cells. 3T3-L1 fat cells were grown to confluence and subjected to different concentrations of tangeritin. The most effective tangeritin inhibition concentration was determined by the MTT assay. The treated cells were subjected to real-time reverse transcriptase PCR and western blot analysis, to detect changes in the CCAAT/enhancer binding protein (C/EBP)α, C/EBPß, and peroxisome proliferator activated receptor (PPAR)γ expression levels. The MTT assay revealed that the fat cell growth was inhibited at a 20 ng/mL concentration of tangeritin. The results of real-time PCR revealed a significant decrease in the expression of C/EBPα, C/EBPß, and PPARγ mRNA, following the treatment with tangeritin. Western blot analysis also presented similar results at a protein level. Therefore, we concluded that tangeritin inhibits adipogenesis via the down-regulation of C/EBPα, C/EBPß, and PPARγ mRNA and protein expression in 3T3-L1 cells.

Molecular Factors Underlying the Deposition of Intramuscular Fat and Collagen in Skeletal Muscle of Nellore and Angus Cattle.

Studies have shown that intramuscular adipogenesis and fibrogenesis may concomitantly occur in skeletal muscle of beef cattle. Thus, we hypothesized that the discrepancy of intramuscular fat content in beef from Nellore and Angus was associated with differences in intramuscular adipogenesis and fibrogenesis during the finishing phase. To test our hypothesis, longissimus muscle samples of Nellore (n = 6; BW = 372.5 ± 37.3 kg) and Angus (n = 6; BW = 382.8 ± 23.9 kg) cattle were collected for analysis of gene and protein expression, and quantification of intramuscular fat and collagen. Least-squares means were estimated for the effect of Breed and differences were considered at P ≤ 0.05. A greater intramuscular fat content was observed in skeletal muscle of Angus compared to Nellore cattle (P≤0.05). No differences were observed for mRNA expression of lipogenic and lipolytic markers ACC, FAS, FABP4, SERBP-1, CPT-2, LPL, and ACOX (P > 0.05) in skeletal muscle of Nellore and Angus cattle. Similarly, no differences were observed in mRNA expression of adipogenic markers Zfp423, PPARγ, and C/EBPα (P>0.05) However, a greater PPARγ protein content was observed in skeletal muscle of Angus compared to Nellore cattle (P≤0.05). A greater abundance of adipo/fibrogenic cells, evaluated by the PDGFRα content, was observed in skeletal muscle of Angus than Nellore cattle (P≤0.05). No differences in fibrogenesis were observed in skeletal muscle of Angus and Nellore cattle, which is in accordance with the lack of differences in intramuscular collagen content in beef from both breeds (P>0.05). These findings demonstrate that difference in intramuscular fat content is associated with a slightly enhanced adipogenesis in skeletal muscle of Angus compared to Nellore cattle, while no difference in fibrogenesis.

C/EBPα gene as a genetic marker for beef quality improvement.

Intramuscular fat (IMF) or intramuscular triglycerides are interspersed throughout the skeletal muscles. The IMF, also called marbling, imparts meat with flavor and juiciness and is one of the core criteria for judging carcass value. The quantity of IMF is influenced entirely by genetics. Recently, understanding the underlying genetic bases of IMF has been a focus particularly in the beef industry. In this study, with the deep insights of ameliorating the beef quality by genetic means, the role of the CCAAT/enhancer binding protein alpha (C/EBPα) gene was investigated by over-expressing C/EBPα in bovine muscle stem cells (MSCs) to initiate the adipogenic program. Prior to this, bovine MSCs were isolated and induced to differentiate into adipocytes from cells that were exposed to dexamethasone isobutylmethylxanthine and indomethacin; the presence of insulin and fetal bovine serum was examined. Either ectopic expression of C/EBPα or treatment with dexamethasone and insulin induced the accumulation of fat droplets and the expression of adipogenic induction genes (LPL, PPARγ, C/EBPß, and C/EBPδ). The expression levels of myoblast-related genes (MyoD, Myf5, and Pax7) were also measured to assess the accuracy of the differentiation process. This study provides evidence that the C/EBPα gene is essential for cattle adipose tissue growth and development. Hence, this finding can contribute to improving beef carcass quality.

Maternal overnutrition enhances mRNA expression of adipogenic markers and collagen deposition in skeletal muscle of beef cattle fetuses.

Twenty-four pregnant Nellore cows were randomly assigned into 2 feeding level groups (control [CTL]; fed 1.0 times the maintenance requirement; n = 12; and overnourished [ON]; fed at 1.5 times the maintenance requirement; n = 12) to evaluate effects of maternal overnutrition on fetal skeletal muscle development. Cows were slaughtered at 135, 190, and 240 d of gestation and samples of fetal LM were collected for analysis of mRNA expression analysis and for histological evaluation of collagen content and number of muscle cells. There was no interaction between gestational period and maternal nutrition for the variables evaluated (P > 0.05). The mRNA expression of Cadherin-associated protein, ß 1 (ß-catenin) tended to be greater in fetuses from ON cows (P = 0.08), while myogenic differentiation 1 (MyoD; P = 0.56), myogenin (MyoG; P = 0.70), and the number of muscle cells (P = 0.90) were not affected by maternal overnutrition. Gestational period did not affect the mRNA expression of ß-catenin (P = 0.60) and MyoG (P = 0.21). The mRNA expression of MyoD tended to increase with days of gestation (P = 0.06). The mRNA expression of zinc finger protein 423 (Zfp423; P < 0.0001), C/EBPα (P = 0.01), and PPARγ (P < 0.0001) were enhanced in ON fetuses. No effects of days of gestation were observed for mRNA expression of Zfp423 (P = 0.75) and C/EBPα (P = 0.48). The mRNA expression of PPARγ in fetuses at 190 d of gestation tended to be greater than those at 135 and 240 d of gestation (P = 0.06). The mRNA expression of transforming growth factor ß (TGF-ß; P < 0.0001), collagen type III, α I (COL3A1; P < 0.0001), and collagen content (P = 0.01) were increased in ON fetuses. Gestational period did not affect the mRNA expression of collagen type I, α I (COL1A1; P = 0.65). The mRNA expression of COL3A1 (P = 0.09) in fetuses at 190 d of gestation tended to be greater than fetuses at 135 and 240 d of gestation. The mRNA expression of TGF-ß in fetuses at 190 d of gestation was greater than in fetuses at 135 d of gestation (P = 0.03), and the values observed in fetuses at 240 d of gestation did not differ from the other gestational time points. The least value of collagen content (P = 0.01) was observed in fetuses at 135 d of gestation, and no differences were observed among the other gestational time points. These data shows that maternal overnutrition enhances fibrogenesis and likely adipogenesis without compromising myogenesis in fetal skeletal muscle of cattle.

C/EBPα is required for long-term self-renewal and lineage priming of hematopoietic stem cells and for the maintenance of epigenetic configurations in multipotent progenitors.

Transcription factors are key regulators of hematopoietic stem cells (HSCs) and act through their ability to bind DNA and impact on gene transcription. Their functions are interpreted in the complex landscape of chromatin, but current knowledge on how this is achieved is very limited. C/EBPα is an important transcriptional regulator of hematopoiesis, but its potential functions in HSCs have remained elusive. Here we report that C/EBPα serves to protect adult HSCs from apoptosis and to maintain their quiescent state. Consequently, deletion of Cebpa is associated with loss of self-renewal and HSC exhaustion. By combining gene expression analysis with genome-wide assessment of C/EBPα binding and epigenetic configurations, we show that C/EBPα acts to modulate the epigenetic states of genes belonging to molecular pathways important for HSC function. Moreover, our data suggest that C/EBPα acts as a priming factor at the HSC level where it actively promotes myeloid differentiation and counteracts lymphoid lineage choice. Taken together, our results show that C/EBPα is a key regulator of HSC biology, which influences the epigenetic landscape of HSCs in order to balance different cell fate options.

Cloning, expression analysis and sequence prediction of the CCAAT/enhancer-binding protein alpha gene of Qinchuan cattle.

CCAAT/enhancer-binding protein alpha (C/EBPα) is an essential transcription factor, regulating the differentiation of adipocytes. We cloned the complete open reading frame of C/EBPα gene of Qinchuan cattle and analyzed its protein structures and expression profile in 15 tissues via DNA cloning, sequencing and RT-PCR. Analysis of the putative protein sequences revealed that C/EBPα consists of alpha helices, random coils and a few extended strands. A significant transmembrane structure was observed in amino acid region 233 to 252. A basic leucine zipper domain was also found in amino acid region 277 to 340, which is characteristic of C/EBPs. Homologous comparison with various species indicated that the C/EBPα gene of Qinchuan cattle shares 97, 95, 94, 94, and 93% similarity in amino acid sequences with Sus scrofa, Homo sapiens, Rattus norvegicus, Oryctolagus cuniculus, and Mus musculus, respectively, implying strong sequence conservation of C/EBPα during evolution. RT-PCR revealed that the mRNA expression level of bovine C/EBPα gene in subcutaneous fat is much higher than that in the other 14 tissues, and the relative quantity in fat tissue increases with cattle age.

Phenolic compounds from maté (Ilex paraguariensis) inhibit adipogenesis in 3T3-L1 preadipocytes.

Leaves of Ilex paraguariensis are used to prepare a tea known as maté which is a common beverage in several South American countries. The ethanol extract was fractionated to identify the compounds responsible for the anti-adipogenic activity in 3T3-L1 cells. Extracts of both fresh and dried maté leaves were subjected to column chromatography using molecular permeation to obtain the saponin (20 % yields) and the polyphenol extracts (40 % yields) from the fresh and dried leaves. The phenolic content was determined using high-performance liquid chromatography analysis and the Folin-Ciocalteau method. Also, maté extracts (50 µg/ml to 1,000 µg/ml) did not display citotoxicity using MTT. The polyphenol extract from the dried leaves was the most effective (50 µg/ml) in the inhibition of triglyceride accumulation in 3T3-L1 adipocytes, and rutin (100 µg/ml) likely accounted for a large portion of this activity. Additionally, maté extracts had a modulatory effect on the expression of genes related to the adipogenesis as PPARγ2, leptin, TNF-α and C/EBPα.

A novel SNP of the C/EBPα gene associated with superior meat quality in indigenous Chinese cattle.

CCAAT/enhancer-binding protein alpha (C/EBPα) is an essential transcriptional factor regulating the differentiation of adipocytes. We report a novel single nucleotide polymorphism (C271A) of the C/EBPα gene in six indigenous Chinese cattle breeds using PCR-SSCP and DNA sequencing methods. Allele frequencies were investigated and evaluated by the χ(2) test in 817 individuals; all populations were found to be in Hardy-Weinberg equilibrium. Gene heterozygosity, effective allele numbers and polymorphism information content of the C/EBPα locus varied from 0.50 to 0.54, 1.84 to 1.99 and 0.35 to 0.37, respectively. We also evaluated a potential association of the C/EBPα SNP with ultrasound traits in 555 individuals; individuals of the AA genotype had greater ultrasound backfat thickness than did genotype CC (0.36 versus 0.34 cm, P < 0.01); genotypes AA and CA had higher ultrasound marbling scores than did genotype CC (3.53, 3.52 versus 3.37, P < 0.05). Analysis based on meat quality data in another 204 Qinchuan cattle showed that animals with genotype AA had bigger loin eye areas than did genotype CA (87.10 versus 79.08 cm(2), P < 0.05). These results indicate that the C271A SNP of the C/EBPα gene could be used as a molecular marker for selecting beef cattle with superior carcass traits.

The CEBPA gene is down-regulated in acute promyelocytic leukemia and its upstream promoter, but not the core promoter, is highly methylated.

Impairment of CCAAT Enhancer Binding Protein alpha (CEBPA) function is a common finding in acute myeloid leukemia; nevertheless, its relevance for acute promyelocytic leukemia pathogenesis is unclear. We analyzed the expression and assessed the methylation status of the core and upstream promoters of CEBPA in acute promyelocytic leukemia at diagnosis. Patients with acute promyelocytic leukemia (n = 18) presented lower levels of CEBPA expression compared to healthy controls (n = 5), but higher levels than those in acute myeloid leukemia with t(8;21) (n = 9) and with inv(16) (n = 5). Regarding the core promoter, we detected no methylation in 39 acute promyelocytic leukemia samples or in 8 samples from controls. In contrast, analysis of the upstream promoter showed methylation in 37 of 39 samples, with 17 patients showing methylation levels over 30%. Our results corroborate data obtained in animal models showing that CEBPA is down-regulated in acute promyelocytic leukemia stem cells and suggest that epigenetic mechanisms may be involved.

Nitric oxide reduces SLC29A1 promoter activity and adenosine transport involving transcription factor complex hCHOP-C/EBPalpha in human umbilical vein endothelial cells from gestational diabetes.

AIMS: Reduced expression of human equilibrative nucleoside transporter 1 (hENT1) results from nitric oxide (NO)-dependent reduced SLC29A1 transcriptional activity in human umbilical vein endothelial cells (HUVECs) from gestational diabetes. As expression of the transcription factor C/EBP homologous protein 10 (hCHOP, which forms heterodimers with C/EBPalpha transcription factor) is activated by NO and induced in diabetes mellitus, we hypothesize that hCHOP plays a role in the gestational diabetes-reduced hENT1 expression in HUVECs. METHODS AND RESULTS: HUVEC primary cultures from 42 normal and 42 gestational diabetic pregnancies were used for adenosine uptake assays. Real-time PCR (mRNA quantification), western blotting (protein abundance), and luciferase activity (SLC29A1 promoter activity) were used. hCHOP-C/EBPalpha activity was assayed by chromatin immunoprecipitation. Overlap extension mutagenesis was used to generate a mutated hCHOP-C/EBPalpha consensus site at the SLC29A1 promoter, and endothelial NO synthase (eNOS) siRNA recombinant adenovirus was used to knock down eNOS. hCHOP nuclear protein abundance and binding to DNA were higher in gestational diabetes, paralleled by reduced SLC29A1 promoter activity, hENT1 expression, and transport activity. These changes were blocked by hCHOP consensus sequence mutation (-1845G > T and -1844C > A), eNOS-siRNA-induced knockdown, and N(G)-nitro-L-arginine methyl ester (NOS inhibitor), and were mimicked by S-nitroso-N-acetyl-L, D-penicillamine (NO donor) in cells from normal pregnancies. hCHOP and C/EBPalpha overexpression mimicked gestational diabetes effects in cells from normal pregnancies, but did not alter SLC29A1 promoter activity or hENT1-adenosine transport in cells from gestational diabetes. CONCLUSION: The hCHOP-C/EBPalpha complex down-regulates SLC29A1 expression in an NO-dependent manner in HUVECs from gestational diabetes.

Adipocyte differentiation is inhibited by melatonin through the regulation of C/EBPbeta transcriptional activity.

Considering that melatonin has been implicated in body weight control, this work investigated whether this effect involves the regulation of adipogenesis. 3T3-L1 preadipocytes were induced to differentiate in the absence or presence of melatonin (10(-3) m). Swiss-3T3 cells ectopically and conditionally (Tet-off system) over-expressing the 34 kDa C/EBPbeta isoform (Swiss-LAP cells) were employed as a tool to assess the mechanisms of action at the molecular level. Protein markers of the adipogenic phenotype were analyzed by Western blot. At 36 hr of differentiation of 3T3-L1 preadipocytes, a reduction of PPARgamma expression was detected followed by a further reduction, at day 4, of perilipin, aP2 and adiponectin protein expression in melatonin-treated cells. Real-time PCR analysis also showed a decrease of PPARgamma (60%), C/EBPalpha (75%), adiponectin (30%) and aP2 (40%) mRNA expression. Finally, we transfected Swiss LAP cells with a C/EBPalpha gene promoter/reporter construct in which luciferase expression is enhanced in response to C/EBPbeta activity. Culture of such transfected cells in the absence of tetracycline led to a 2.5-fold activation of the C/EBPalpha promoter. However, when treated with melatonin, the level of C/EBPalpha promoter activation by C/EBPbeta was reduced by 50% (P = 0.05, n = 6). In addition, this inhibitory effect of melatonin was also reflected in the phenotype of the cells, since their capacity to accumulate lipids droplets was reduced as confirmed by the poor staining with Oil Red O. In conclusion, melatonin at a concentration of 10(-3 ) m works as a negative regulator of adipogenesis acting in part by inhibiting the activity of a critical adipogenic transcription factor, C/EBPbeta.