FBP1 Expression Research Articles

Trehalose-6-phosphate synthesis controls yeast gluconeogenesis downstream and independent of SNF1.

Trehalose-6-P (T6P), an intermediate of trehalose biosynthesis, was identified as an important regulator of yeast sugar metabolism and signaling. tps1Δ mutants, deficient in T6P synthesis (TPS), are unable to grow on rapidly fermentable medium with uncontrolled influx in glycolysis, depletion of ATP and accumulation of sugar phosphates. However, the exact molecular mechanisms involved are not fully understood. We show that SNF1 deletion restores the tps1Δ growth defect on glucose, suggesting that lack of TPS hampers inactivation of SNF1 or SNF1-regulated processes. In addition to alternative, non-fermentable carbon metabolism, SNF1 controls two major processes: respiration and gluconeogenesis. The tps1Δ defect appears to be specifically associated with deficient inhibition of gluconeogenesis, indicating more downstream effects. Consistently, Snf1 dephosphorylation and inactivation on glucose medium are not affected, as confirmed with an in vivo Snf1 activity reporter. Detailed analysis shows that gluconeogenic Pck1 and Fbp1 expression, protein levels and activity are not repressed upon glucose addition to tps1Δ cells, suggesting a link between the metabolic defect and persistent gluconeogenesis. While SNF1 is essential for induction of gluconeogenesis, T6P/TPS is required for inactivation of gluconeogenesis in the presence of glucose, downstream and independent of SNF1 activity and the Cat8 and Sip4 transcription factors.

Down-regulation of FBP1 by ZEB1-mediated repression confers to growth and invasion in lung cancer cells.

Lung cancer is the most common type of malignant tumor, but the molecular mechanisms for lung cancer progression remains to be elusive. Here, we demonstrated that FBP1 (Fructose-1, 6-bisphosphatase) was frequently down-regulated in lung cancer tissues and cells, and FBP1 down-regulation was associated with poor prognosis in lung cancer patients. Restored FBP1 expression inhibited glucose uptake and lactate production, but induced oxygen consumption. Restored FBP1 expression also inhibited lung cancer cells proliferation and invasion under hypoxia in vitro, and inhibited lung cancer growth in vivo. Moreover, we confirmed DNA methylation in the promoter contributed to the decrease of FBP1 expression in lung cancer cells. We identified Zinc finger E-box-binding homeobox 1 (ZEB1) bond to FBP1 promoter to enhance DNA methylation in lung cancer cells. Our findings indicate that the down-regulation of FBP1 is a critical oncogenic event in lung cancer progression.

AB180. The relationship between fructose-1, 6-bisphosphatase and hypoxia related genes expression in clear cell renal cell carcinoma

ObjectiveFructose-1,6-bisphosphatase (FBP1) is often known as a rate-limiting enzyme in gluconeogenesis. Recently, its catalytic activity-independent function, repress hypoxia induced factor (HIF) in the nucleus, was identified. The aim of this study was to investigate the relationship between FBP1 and hypoxia related genes expression in clear cell renal cell carcinoma (ccRCC).MethodsThe expression levels of FBP1, HIF-1α, HIF-2α, erythropoietin (Epo) and carbonic anhydrase IX (CA9) were assessed by immunochemical staining in archival ccRCC paraffin blocks from 123 patients using the tissue microarray technique. The expression level of FBP1 was then correlated with clinicopathological factors and the expression levels of HIF-1α, HIF-2α, Epo and CA9.ResultsClinicopathological factors including age, gender, TNM stage and Fuhrman grade were indifferent between the patients with low FBP1 expression and those with strong FBP1 expression in ccRCC. FBP1 expression level was positively correlated with the expression levels of HIF-1α (P=0.005) and Epo (P=0.010), but without correlation with the expression level of HIF-2α (P=0.123) and CA9 (P=0.513) in ccRCC tissues.ConclusionsOur findings may be useful for recognizing the association between FBP1 and hypoxia related genes expression and understanding the mechanisms of ccRCC tumorigenesis.

Fasting for 3 days during the suckling-weaning transient period in male rats induces metabolic abnormalities in the liver and is associated with impaired glucose tolerance in adulthood.

Recent studies suggest that nutritional status during developmental periods is associated with subsequent development of metabolic abnormalities. In this study, we examined whether malnutrition by fasting for 3 days during the suckling-weaning transient period induces subsequent development of metabolic abnormalities in rats. Male Sprague-Dawley rats were fasted for 3 days during the suckling-weaning transient period. They are subsequently fed a high-fat, high-sucrose (HF) or low-fat, high-starch (LF) diet for 14 weeks from 17 weeks of age, and the liver and blood samples were collected for measuring mRNA and protein levels of metabolic genes and blood concentrations of glucose and insulin, respectively. Fasting for 3 days during the suckling-weaning transient period induced impaired glucose tolerance in rats fed the LF diet in adulthood. Liver triglycerides in rats fed the HF diet in adulthood increased to 140 % in rats fasted for 3 days during the suckling-weaning transient period compared with those non-fasted. Furthermore, liver expression of FBP1 and ACCα genes in adult rats fed the LF diet increased to 125 and 145 %, respectively, in rats fasted for 3 days during the suckling-weaning transient period compared to non-fasted rats. PEPCK1 protein expression levels in rats fed the LF diet were higher in rats fasted for 3 days during the suckling-weaning transient period than in non-fasted rats. Fasting for 3 days in rats during the suckling-weaning transient period enhances metabolic abnormalities in animals fed a HF or LF diet in adulthood by confounding metabolism of lipid and sugar in the liver.

Expression of far upstream element (FUSE) binding protein 1 in human glioma is correlated with c‐Myc and cell proliferation

Glioma is one of the most common type of primary intracranial tumor. Although great advances have been achieved in treatment of glioma, the underlying molecular mechanisms remain largely unknown. Previous studies demonstrated that FBP1 is a transcriptional regulator of c-Myc and acts as an important prognostic indicator in many cancers. Our study aimed to assess the expression and function of FBP1 in human glioma. Immunohistochemical and Western blot analysis were performed in human glioma and normal brain tissues. High FBP1 expression (located in cell nuclei) was observed in 70 samples and its level was correlated with the grade of malignancy. A strongly positive correlation was observed between FBP1 and c-Myc (P = 0.005) and Ki-67 expression (P = 0.009). In a multivariate analysis, high FBP1 and c-Myc expressions were showed to be associated with poor prognosis in glioma. While in vitro, following serum stimulation of starved U87MG cells, the expression of FBP1 was upregulated, as well as c-Myc and PCNA. Moreover, knockdown of FBP1 by siRNA transfection diminished the expression of c-Myc and arrested cell growth at G1 phase. Collectively, our results shows that the expression of FBP1 is in close correlation with c-Myc level and cell proliferation in glioma and provides a potential strategy to develop FBP1 inhibitors as novel anti-tumor agents.

The Expression of FBP1 after Traumatic Brain Injury and Its Role in Astrocyte Proliferation

Far upstream element binding protein 1 (FBP1) has been identified as an upstream gene of p27kip1 (p27), which is a key regulator of mammalian cell cycle regulation and neurogenesis. To elucidate the expression and function of FBP1 in central nervous system lesion and repair, we performed a traumatic brain injury (TBI) model in adult rats. We observed that FBP1 protein level significantly reduced at day 3 after injury, and the downregulation of FBP1 was predominant in astrocytes, which were largely proliferated after injury. Furthermore, in vitro, overexpression of FBP1 was concomitant with the up-regulation of p27 and reduction of PCNA in LPS-induced astrocyte proliferation. These results suggest that a decreased level of FBP1 in brain is involved in the proliferation of glial cells after TBI.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Role of the fission yeast cell integrity MAPK pathway in response to glucose limitation

BackgroundGlucose is a signaling molecule which regulates multiple events in eukaryotic organisms and the most preferred carbon source in the fission yeast Schizosaccharomyces pombe. The ability of this yeast to grow in the absence of glucose becomes strongly limited due to lack of enzymes of the glyoxylate cycle that support diauxic growth. The stress-activated protein kinase (SAPK) pathway and its effectors, Sty1 MAPK and transcription factor Atf1, play a critical role in the adaptation of fission yeast to grow on alternative non-fermentable carbon sources by inducing the expression of fbp1+ gene, coding for the gluconeogenic enzyme fructose-1,6-bisphosphatase. The cell integrity Pmk1 pathway is another MAPK cascade that regulates various processes in fission yeast, including cell wall construction, cytokinesis, and ionic homeostasis. Pmk1 pathway also becomes strongly activated in response to glucose deprivation but its role during glucose exhaustion and ensuing adaptation to respiratory metabolism is currently unknown.ResultsWe found that Pmk1 activation in the absence of glucose takes place only after complete depletion of this carbon source and that such activation is not related to an endogenous oxidative stress. Notably, Pmk1 MAPK activation relies on de novo protein synthesis, is independent on known upstream activators of the pathway like Rho2 GTPase, and involves PKC ortholog Pck2. Also, the Glucose/cAMP pathway is required operative for full activation of the Pmk1 signaling cascade. Mutants lacking Pmk1 displayed a partial growth defect in respiratory media which was not observed in the presence of glucose. This phenotype was accompanied by a decreased and delayed expression of transcription factor Atf1 and target genes fbp1+ and pyp2+. Intriguingly, the kinetics of Sty1 activation in Pmk1-less cells was clearly altered during growth adaptation to non-fermentable carbon sources.ConclusionsUnknown upstream elements mediate Pck2-dependent signal transduction of glucose withdrawal to the cell integrity MAPK pathway. This signaling cascade reinforces the adaptive response of fission yeast to such nutritional stress by enhancing the activity of the SAPK pathway.

Abstract 1300: Far upstream element binding proteins (FBP)-1 and FBP-2 in non-small cell lung cancer: Key factors regulating tumor cell proliferation, migration, and invasion

Abstract The far upstream element (FUSE)-binding proteins (FBP)- 1, FBP-2, and FBP-3 represent a family of single-strand nucleic acid binding factors, regulating transcriptional as well as post-transcriptional processes. Elevated expression and pro-tumorigenic functions of all FBPs have been described for human liver cancer [1, 2]. First data indicated that FBP-1 affects microtubule dynamics through regulating MT-destabilizing factors in non-small cell lung cancer (NSCLC) [3], however, comprehensive studies analyzing the expression and functional relevance of all FBPs in NSCLCs are missing so far. In order to define the expression of FBPs in lung cancer, FBP-transcript and protein levels were analysed in primary human NSCLC tissue samples. Semiquantitative real-time PCR as well as Tissue-Micro-Array (TMA) analyses revealed an elevated expression of FBP-1 and FBP-2 in most NSCLC tissue samples (>60%) in comparison to non-tumorous specimens. Interestingly, nuclear accumulation of FBP-1 significantly correlated with FBP-2 expression (r=0.33; p<0.01), suggesting common regulatory mechanisms. In vitro, transient inhibition of FBP-1 by gene-specific siRNAs in NSCLC cell lines (Calu-6) was associated with decreased tumor cell viability (-76%; MTT assay), proliferation (-83%; BrdU assay), and increased apoptosis (2.8-fold; Nicoletti FACS assay). In contrast, inhibition of FBP-2 reduced cell viability of Calu-1 cells (-32%; MTT assay) but predominantly reduced tumor cell migration (-62%; two-dimensional scratch assay) as well as tumor cell invasion (-81%; sprouting assay) in all analyzed NSCLC cell lines (Calu-1, Calu-6, and A549), suggesting that both FBP isoforms facilitate partly distinct tumor-supporting effects. Surprisingly, efficient single-gene inhibition of FBP-1 or FBP-2 in A549 cells did not affect tumor cell viability. In contrast, the concomitant knock down of both FBP isoforms resulted in significantly decreased cell viability (-69%), suggesting that some FBP family members may compensate the loss of other members. Actually, FBP-2 negatively regulates FBP-1 expression in A549 cells, resulting in increased FBP-1 transcript and protein levels after FBP-2 inhibition. Therefore, functional compensation prevents A549 cells from anti-tumorigenic effects after FBP-2 knock down. In summary, this study provides evidence that coordinated overexpression of FBP-1 and FBP-2 is a frequent event in NSCLCs and that both factors are support tumor growth and NSCLC cell dissemination. Interestingly, partial functional redundance and mutual negative regulation of FBPs indicate that these factors may fine-tune the oncogenic behavior of NSCLC cells.

The Zinc Finger Protein ZBTB20 Regulates Transcription of Fructose-1,6-Bisphosphatase 1 and β Cell Function in Mice

Fructose-1,6-bisphosphatase (FBP)-1 is a gluconeogenic enzyme that regulates glucose metabolism and insulin secretion in β cells, but little is known about how its transcription is controlled. The zinc finger protein ZBTB20 regulates glucose homeostasis, so we investigated its effects on expression of FBP-1. We analyzed gene expression using real-time reverse-transcription polymerase chain reaction, immunoblotting, and immunohistochemistry. We generated mice with β cell-specific disruption of Zbtb20 using Cre/LoxP technology. Expression of Zbtb20 in β cells was reduced using small interfering RNAs, and promoter occupancy and transcriptional regulation were analyzed by chromatin immunoprecipitation and reporter assays. ZBTB20 was expressed at high levels by β cells and other endocrine cells in islets of normal mice; expression levels were reduced in islets from diabetic db/db mice. Mice with β cell-specific knockout of Zbtb20 had normal development of β cells but had hyperglycemia, hypoinsulinemia, glucose intolerance, and impaired glucose-stimulated insulin secretion. Islets isolated from these mice had impaired glucose metabolism, adenosine triphosphate production, and insulin secretion after glucose stimulation in vitro, although insulin secretion returned to normal levels in the presence of KCl. ZBTB20 knockdown with small interfering RNAs impaired glucose-stimulated insulin secretion in the β cell line MIN6. Expression of Fbp1 was up-regulated in β cells with ZBTB20 knockout or knockdown; impairments to glucose-stimulated insulin secretion were restored by inhibition of FBPase activity. ZBTB20 was recruited to the Fbp1 promoter and repressed its transcription in β cells. The transcription factor ZBTB20 regulates β cell function and glucose homeostasis in mice. It might be a therapeutic target for type 2 diabetes mellitus.

Promoter Hypermethylation Mediated Downregulation of FBP1 in Human Hepatocellular Carcinoma and Colon Cancer

FBP1, fructose-1,6-bisphosphatase-1, a gluconeogenesis regulatory enzyme, catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. The mechanism that it functions to antagonize glycolysis and was epigenetically inactivated through NF-kappaB pathway in gastric cancer has been reported. However, its role in the liver carcinogenesis still remains unknown. Here, we investigated the expression and DNA methylation of FBP1 in primary HCC and colon tumor. FBP1 was lowly expressed in 80% (8/10) human hepatocellular carcinoma, 66.7% (6/9) liver cancer cell lines and 100% (6/6) colon cancer cell lines, but was higher in paired adjacent non-tumor tissues and immortalized normal cell lines, which was well correlated with its promoter methylation status. Methylation was further detected in primary HCCs, gastric and colon tumor tissues, but none or occasionally in paired adjacent non-tumor tissues. Detailed methylation analysis of 29 CpG sites at a 327-bp promoter region by bisulfite genomic sequencing confirmed its methylation. FBP1 silencing could be reversed by chemical demethylation treatment with 5-aza-2′-deoxycytidine (Aza), indicating direct epigenetic silencing. Restoring FBP1 expression in low expressed cells significantly inhibited cell growth and colony formation ability through the induction of G2-M phase cell cycle arrest. Moreover, the observed effects coincided with an increase in reactive oxygen species (ROS) generation. In summary, epigenetic inactivation of FBP1 is also common in human liver and colon cancer. FBP1 appears to be a functional tumor suppressor involved in the liver and colon carcinogenesis.

Chromatin occupancy of transcription factor 7-like 2 (TCF7L2) and its role in hepatic glucose metabolism

The mechanisms by which transcription factor 7-like 2 (TCF7L2) regulates the pathways that are important in the pathogenesis of type 2 diabetes are unknown. We therefore examined the role of TCF7L2 in hepatic glucose production (HGP) in vitro and characterised the whole-genome chromatin occupancy of TCF7L2 in hepatocytes. We investigated the effect of TCF7L2 silencing and overexpression on HGP from gluconeogenic precursors and used chromatin-immunoprecipitation (ChIP) combined with massively parallel DNA sequencing (ChIP-Seq) to investigate the DNA binding patterns of TCF7L2 across the whole genome. Silencing of TCF7L2 induced a marked increase in basal HGP, which was accompanied by significant increases in the expression of the gluconeogenic genes Fbp1, Pck1 and G6pc. Overexpression of Tcf7l2 reversed this phenotype and significantly reduced HGP. TCF7L2 silencing did not affect the half-maximal inhibitory concentration of insulin or metformin, but HGP remained elevated in TCF7L2-silenced cells due to the increased baseline HGP. Using ChIP-Seq, we detected 2,119 binding events across the genome. Pathway analysis demonstrated that diabetes genes were significantly over-represented in the dataset. Our results indicate that TCF7L2 binds directly to multiple genes that are important in regulation of glucose metabolism in the liver, including Pck1, Fbp1, Irs1, Irs2, Akt2, Adipor1, Pdk4 and Cpt1a. TCF7L2 is an important regulator of HGP in vitro and binds directly to genes that are important in pathways of glucose metabolism in the liver. These data highlight the possibility that TCF7L2 may affect fasting and postprandial hyperglycaemia in carriers of at-risk TCF7L2 genetic polymorphisms.

Role for PPARγ in obesity‐induced hepatic steatosis as determined by hepatocyte‐ and macrophage‐specific conditional knockouts

Peroxisome proliferator-activated receptor (PPAR) γ is a nuclear receptor central to glucose and lipid homeostasis. PPARγ role in nonalcoholic fatty liver disease is controversial because PPARγ overexpression is a general property of steatotic livers, but its activation by thiazolidinediones reduces hepatic steatosis. Here, we investigated hepatic PPARγ function by using Cre-loxP technology to generate hepatocyte (PPARγ(Δhep))- and macrophage (PPARγ(Δmac))-specific PPARγ-knockout mice. Targeted deletion of PPARγ in hepatocytes, and to a lesser extent in macrophages, protected mice against high-fat diet-induced hepatic steatosis. Down-regulated expression of genes involved in lipogenesis (SCD1, SREBP-1c, and ACC), lipid transport (CD36/FAT, L-FABP, and MTP), and β-oxidation (PPARα and ACO) was observed in PPARγ(Δhep) mice. Moreover, PPARγ(Δhep) mice showed improved glucose tolerance and reduced PEPCK expression without changes in Pcx, Fbp1, and G6Pc expression and CREB and JNK phosphorylation. In precision-cut liver slices (PCLSs) and hepatocytes, rosiglitazone either alone or in combination with oleic acid increased triglyceride accumulation, an effect that was blocked by the PPARγ antagonist biphenol A diglycidyl ether (BADGE). PCLSs and hepatocytes from PPARγ(Δhep) mice showed blunted responses to rosiglitazone and oleic acid, whereas the response to these compounds remained intact in PCLSs from PPARγ(Δmac) mice. Collectively, these findings establish PPARγ expression in hepatocytes as a prosteatotic factor in fatty liver disease.

A proteomic analysis of the functional effects of fatty acids in NIH 3T3 fibroblasts.

Previous studies have demonstrated that long chain fatty acids influence fibroblast function at sub-lethal concentrations. This study is the first to assess the effects of oleic, linoleic or palmitic acids on protein expression of fibroblasts, as determined by standard proteomic techniques. The fatty acids were not cytotoxic at the concentration used in this work as assessed by membrane integrity, DNA fragmentation and the MTT assay but significantly increased cell proliferation. Subsequently, a proteomic analysis was performed using two dimensional difference gel electrophoresis (2D-DIGE) and MS based identification. Cells treated with 50 μM oleic, linoleic or palmitic acid for 24 h were associated with 24, 22, 16 spots differentially expressed, respectively. Among the identified proteins, α-enolase and far upstream element binding protein 1 (FBP-1) are of importance due to their function in fibroblast-associated diseases. However, modulation of α-enolase and FBP-1 expression by fatty acids was not validated by the Western blot technique.

The Elevated Expression of FBP1, a Key-Enzyme of Gluconeogenesis Gene, Is Associated to High Sokal Risk In Chronic Myeloid Leukemia Patients.

AbstractAbstract 3399The oncogenic transformation of Chronic Myeloid Leukemia (CML) cell of origin has been associated to an increased glucose metabolism, where the glycolytic pyruvate is directed away from the mitocondria, converted into lactate and secreted from the cell. This metabolic conversion is termed aerobic glycolysis, or Warburg effect and very essentially, it serves to support the tumor cell proliferation, by providing nucleotides, aminoacids and lipids enough to replicate all of cellular content. CML patients are historically stratified according to their Sokal risk score, which for more than 30 years has been regarded as the most significant prognostic factor in this hematological malignancies. The putative genetic and/or genomic basis driving this stratification are still not known.Aim of the present study was to explore the molecular mechanisms associated to the CML patients stratification according to the Sokal risk score, by analyzing the transcriptome of the CD34+ cell fractions obtained at diagnosis from a cohort of high and non-high Sokal risk CML patients.Overall, 67 patients with previously untreated CML in chronic phase (CP) entered the study; all of them have been enrolled in GIMEMA CML protocols and provided highly enriched CD34+ cell fractions from peripheral blood. Gene expression profiling (GEP) was performed, in order to identify genes most significantly and most differentially expressed between high and non-high Sokal risk patients. All other cases were used in Real-time experiments, in order to validate the GEP data.By GEP, 82 probe-sets, corresponding to 78 genes resulted significantly differentially expressed between high and non-high Sokal risk patients in a supervised analysis of gene profiles. A gene enrichment analysis of this profile showed that genes involved in the Wnt, in the Notch signaling pathways and in the response to hypoxia and oxidative stress resulted significantly overexpressed in the comparison between high and non-high risk patients. We focused our attention on genes involved in the glycolysis and gluconeogenesis metabolic pathways (FBP1, SEPP1, GSTM3, GSTT1, PRDX2, MPO). We validated by Real-time the de-regulated expression of these genes in a different set of newly diagnosed CP-CML patients, thus confirming that they are differentially expressed between high and non-high risk patients, with trends similar to those observed by GEP. Particularly interesting resulted a significantly higher expression in high Sokal risk patients of FBP1 (fructose 1,6 bisphosphatase), a key-enzyme of gluconeogenesis, together with a significant over-expression of genes coding for enzyme involved in gluthatione biosynthesis (GSTM3, GSTT1, and PRDX2). These data suggest that CD34+ cells obtained from high Sokal risk patients might exhibit an unexpected moderation of the glycolytic flux, mainly due to the over-expression of FBP1, which might cause a re-direction of the pathway into the pentose phosphate shunt. A similar metabolic reprogramming has been already described in imatinib resistant bcr-abl positive cell lines and is supported also by the over-expression in high risk patients of G6PDH (Glucose-6-phosphate 1-dehydrogenase) and TK (Transketolase), which are key enzyme of the pentose phosphate shunt.Overall, our data demonstrate for the first time, that the expression at diagnosis of sugar metabolic enzymes, might drive the evolutive Sokal risk of CML patients.Supported by: European LeukemiaNet, BolognAIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrate program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures:Rosti:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Baccarani:NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

In vitro differentiation of unrestricted somatic stem cells into functional hepatic‐like cells displaying a hepatocyte‐like glucose metabolism

The hepatic-like phenotype resulting from in vitro differentiation of unrestricted somatic stem cells (USSC) derived from human umbilical cord blood (CB) was analyzed with regard to functional and metabolic aspects. USSC can be differentiated into cells of all three germ layers in vitro and in vivo and, although they share many features with mesenchymal stroma cells (MSC), can be distinguished from these by their expression of DLK1 as well as a restricted adipogenic differentiation potential. For the differentiation procedure described herein, a novel three-stage differentiation protocol resembling embryonic developmental processes of hepatic endoderm was applied. Hepatic pre-induction was performed by activinA and FGF4 resulting in enhanced SOX17 and FOXA2 expression. Further differentiation was achieved sequentially by retinoic acid, FGF4, HGF, EGF, and OSM resulting in a hepatic endodermal identity, characterized by the expression of AFP and HNF1alpha. Thereafter, expression of G6PC, ARG1, FBP1, and HNF4alpha was observed, thus indicating progressive differentiation. Functional studies concerning albumin secretion, urea formation, and cytochrome-p450-3A4 (CYP3A4) enzyme activity confirmed the hepatic-like phenotype. In order to characterize the differentiated cells at a metabolic level, USSC were incubated with [1-(13)C]glucose. By tracing the fate of the molecule's label via isotopomer analysis using (13)C NMR spectroscopy, formation of both glycogen and some gluconeogenetic activity could be observed providing evidence of a hepatocyte-like glucose metabolism in differentiated USSC. In conclusion, the results of the present study indicate that USSC represent a stem cell source with a substantial hepatic differentiation capacity which hold the potential for clinical applications.

The FUSE binding proteins FBP1 and FBP3 are potential c-myc regulators in renal, but not in prostate and bladder cancer

BackgroundThe three far-upstream element (FUSE) binding proteins (FBP1, FBP2, and FBP3) belong to an ancient family of single-stranded DNA binding proteins which are required for proper regulation of the c-myc proto-oncogene. Whereas it is known that c-myc alterations play a completely different role in various carcinomas of the urogenital tract, the relevance of FBPs is unclear.MethodsFBP1, FBP3 and c-myc expression was studied in 105 renal cell, 95 prostate and 112 urinary bladder carcinomas by immunohistochemistry using tissue microarrays.ResultsHigh rates of FBP1 and FBP3 expression were observed in all cancer types. There was a concomitant up-regulation of FBP1 and FBP3 in renal cell and prostate carcinomas (p < 0.001 both). C-myc expression was detectable in 21% of prostate, 30% of renal and 34% of urothelial carcinomas. Interestingly, strong FBP1 and FBP3 expression was associated with c-myc up-regulation in clear cell renal cell carcinomas (p < 0.001 and 0.09 resp.), but not in bladder or prostate cancer.ConclusionThe correlation between FBP1/FBP3, c-myc and high proliferation rate in renal cell carcinoma provides strong in vivo support for the suggested role of FBP1 and FBP3 as activators of c-myc. The frequent up-regulation of FBP1 and FBP3 in urothelial and prostate carcinoma suggests that FBPs also have an important function in gene regulation of these tumors.

The Transcriptional Coactivators SAGA, SWI/SNF, and Mediator Make Distinct Contributions to Activation of Glucose-repressed Genes

The paradigm of activation via ordered recruitment has evolved into a complicated picture as the influence of coactivators and chromatin structures on gene regulation becomes understood. We present here a comprehensive study of many elements of activation of ADH2 and FBP1, two glucose-regulated genes. We identify SWI/SNF as the major chromatin-remodeling complex at these genes, whereas SAGA (Spt-Ada-Gcn5-acetyltransferase complex) is required for stable recruitment of other coactivators. Mediator plays a crucial role in expression of both genes but does not affect chromatin remodeling. We found that Adr1 bound unaided by coactivators to ADH2, but Cat8 binding depended on coactivators at FBP1. Taken together, our results suggest that commonly regulated genes share many aspects of activation, but that gene-specific regulators or elements of promoter architecture may account for small differences in the mechanism of activation. Finally, we found that activator overexpression can compensate for the loss of SWI/SNF but not for the loss of SAGA.

Spatial and temporal expression of folate-binding protein 1 (Fbp1) is closely associated with anterior neural tube closure in mice.

Periconceptional folate supplementation is widely believed to have significant preventive effects on the production of neural tube defects. Folate-binding protein 1 (FBP1) is one of the membrane proteins that mediate cellular uptake of folate. Although recent studies suggest that Fbp1 is essential for neural tube closure, the pattern of Fbp1 expression in embryonic tissues has not been examined in detail. To elucidate how Fbp1 contributes to neural tube closure, we examined the spatial and temporal expression patterns of Fbp1 in the developing neural folds and tube of mouse embryos by in situ hybridization. Fbp1 showed a distinct expression pattern in the neural folds, which preceded initiation of neural tube closure at the cervical region and the prosencephalic/mesencephalic boundary. Fbp1 expression was mainly localized to the most dorsal regions of the neural folds where fusion was to occur. With proceeding of neural fold fusion, Fbp1 expression extended to the adjacent unfused neural folds. In the rhombencephalon, robust expression of Fbp1 was observed in rhombomere2 (r2) and r6, suggesting its roles in development of neural crest cells. Fbp1 also showed intense expression in the yolk sac, indicating that FBP1 may mediate transferring maternal folate to embryos during neurulation. These findings indicate close association between Fbp1 and anterior neural tube closure.

Elements from the cAMP signaling pathway are involved in the control of expression of the yeast gluconeogenic gene FBP1

cAMP represses the transcription of some Saccharomyces cerevisiae genes sensitive to catabolite repression. The effect of cAMP on the expression of FBP1, encoding fructose-1,6-bisphosphatase (FbPase), has been further investigated. In yeast cells shifted to a derepressing medium, synthesis of FbPase was delayed if the strong decrease in intracellular cAMP, which occurs during the shift, was prevented. A similar delay occurred in a RAS2 val19 strain, while in a tpk1 w strain, with weak protein kinase A activity, induction of FbPase occurred earlier than in a TPK1 strain. In the tpk1 w strain, proteins which bind the UAS1 element of FBP1 were present during growth on glucose but they were only weakly operative. Expression of CAT8 and SIP4, encoding proteins which bind the UAS2 element, was blocked by a high concentration of cAMP, but catabolite repression of these genes was not much relieved in a tpk1 w strain. We conclude that in S. cerevisiae, as reported for Schizosaccharomyces pombe, control of FBP1 requires both cAMP-dependent and independent pathways; however, the mechanisms operating in the two yeasts are different.