BCL-6: a possible missing link for anti-inflammatory PPAR-delta signalling in pancreatic beta cells.

AIMS/HYPOTHESIS: Inflammatory mediators contribute to pancreatic beta cell death in type 1 diabetes. Beta cells respond to cytokine exposure by activating gene networks that alter cellular metabolism, induce chemokine release (thereby increasing insulitis), and cause apoptosis. We have previously shown by microarray analysis that exposure of INS-1E cells to IL-1beta + IFN-gamma induces the transcription factor peroxisome proliferator-activated receptor (Ppar)-delta and several of its target genes. PPAR-delta controls cellular lipid metabolism and is a major regulator of inflammatory responses. We therefore examined the role of PPAR-delta in cytokine-treated beta cells. MATERIALS AND METHODS: Primary beta cells that had been purified by fluorescence-activated cell sorting and INS-1E cells were cultured in the presence of the cytokines TNF-alpha, IL-1beta, or IL-1beta + IFN-gamma, or the synthetic PPAR-delta agonist GW501516. Gene expression was analysed by real-time PCR. PPAR-delta, monocyte chemoattractant protein (MCP-1, now known as CCL2) promoter and NF-kappaB activity were determined by luciferase reporter assays. RESULTS: Exposure of primary beta cells or INS-1E cells to cytokines induced Ppar-delta mRNA expression and PPAR-delta-dependent CD36, lipoprotein lipase, acyl CoA synthetase and adipophilin mRNAs. Cytokines and the PPAR-delta agonist GW501516 also activated a PPAR-delta response element reporter in beta cells. Unlike immune cells, neither INS-1E nor beta cells expressed the transcriptional repressor B-cell lymphoma-6 (BCL-6). As a consequence, PPAR-delta activation by GW501516 did not decrease cytokine-induced Mcp-1 promoter activation or mRNA expression, as reported for macrophages. Transient transfection with a BCL-6 expression vector markedly reduced Mcp-1 promoter and NF-kappaB activities in beta cells. CONCLUSIONS/INTERPRETATION: Cytokines activate the PPAR-delta gene network in beta cells. This network does not, however, regulate the pro-inflammatory response to cytokines because beta cells lack constitutive BCL-6 expression. This may render beta cells particularly susceptible to propagating inflammation in type 1 diabetes.

Extracellular signal-regulated kinase is essential for interleukin-1-induced and nuclear factor kappaB-mediated gene expression in insulin-producing INS-1E cells.

AIMS/HYPOTHESIS: The beta cell destruction and insulin deficiency that characterises type 1 diabetes mellitus is partially mediated by cytokines, such as IL-1beta, and by nitric oxide (NO)-dependent and -independent effector mechanisms. IL-1beta activates mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK), and the nuclear factor kappa B (NFkappaB) pathway. Both pathways are required for expression of the gene encoding inducible nitric oxide synthase (iNOS) and for IL-1beta-mediated beta cell death. The molecular mechanisms by which these two pathways regulate beta cell Nos2 expression are currently unknown. Therefore, the aim of this study was to clarify the putative crosstalk between MAPK and NFkappaB activation in beta cells. MATERIALS AND METHODS: The MAPKs ERK, p38 and JNK were inhibited by SB203580, PD98059 or Tat-JNK binding domain or by cells overexpressing the JNK binding domain. The effects of MAPK inhibition on IL-1beta-induced iNOS production and kappa B inhibitor protein (IkappaB) degradation were examined by western blotting. NFkappaB DNA binding was investigated by electrophoretic mobility shift assay, while NFkappaB-induced gene transcription was evaluated by gene reporter assays. RESULTS: Inhibition of the MAPKs did not affect IkappaB degradation or NFkappaB DNA binding. However, inhibition of ERK reduced NFkappaB-mediated Nos2 expression; serine 276 phosphorylation of the p65 unit of the NFkappaB complex seemed critical, as evaluated by amino acid mutation analysis. CONCLUSIONS/INTERPRETATION: ERK activity is required for NFkappaB-mediated transcription of Nos2 in insulin-producing INS-1E cells, indicating that ERK regulates Nos2 expression by increasing the transactivating capacity of NFkappaB. This may involve phosphorylation of Ser276 on p65 by an as yet unidentified kinase.

Suppressor of cytokine signalling (SOCS)-3 protects beta cells against IL-1beta-mediated toxicity through inhibition of multiple nuclear factor-kappaB-regulated proapoptotic pathways.

AIMS/HYPOTHESIS: The proinflammatory cytokine IL-1beta induces apoptosis in pancreatic beta cells via pathways dependent on nuclear factor-kappaB (NF-kappaB), mitogen-activated protein kinase, and protein kinase C. We recently showed suppressor of cytokine signalling (SOCS)-3 to be a natural negative feedback regulator of IL-1beta- and IFN-gamma-mediated signalling in rat islets and beta cell lines, preventing their deleterious effects. However, the mechanisms underlying SOCS-3 inhibition of IL-1beta signalling and prevention against apoptosis remain unknown. METHODS: The effect of SOCS-3 expression on the global gene-expression profile following IL-1beta exposure was microarray-analysed using a rat beta cell line (INS-1) with inducible SOCS-3 expression. Subsequently, functional analyses were performed. RESULTS: Eighty-two known genes and several expressed sequence tags (ESTs) changed expression level 2.5-fold or more in response to IL-1beta alone. Following 6 h of IL-1beta exposure, 23 transcripts were up-regulated. Of these, several, including all eight transcripts relating to immune/inflammatory response pathways, were suppressed by SOCS-3. Following 24 h of IL-1beta exposure, secondary response genes were detected, affecting metabolism, energy generation, protein synthesis and degradation, growth arrest, and apoptosis. The majority of these changes were prevented by SOCS-3 expression. Multiple IL-1beta-induced NF-kappaB-dependent proapoptotic early response genes were inhibited by SOCS-3 expression, suggesting that SOCS-3 inhibits NF-kappaB-mediated signalling. These observations were experimentally confirmed in functional analyses. CONCLUSIONS/INTERPRETATION: This study suggests that there is an unexpected cross-talk between the SOCS/IFN and the IL-1beta pathways of signalling in pancreatic beta cells, which could lead to a novel perspective of blocking two important proapoptotic pathways in pancreatic beta cells by influencing a single signalling molecule, namely SOCS-3.

Inhibition of cytokine-induced NF-kappaB activation by adenovirus-mediated expression of a NF-kappaB super-repressor prevents beta-cell apoptosis.

Cytokine-induced beta-cell death is an important event in the pathogenesis of type 1 diabetes. The transcription factor nuclear factor-kappaB (NF-kappaB) is activated by interleukin-1beta (IL-1beta), and its activity promotes the expression of several beta-cell genes, including pro- and anti-apoptotic genes. To elucidate the role of cytokine (IL-1beta + gamma-interferon [IFN-gamma])-induced expression of NF-kappaB in beta-cell apoptosis, rat beta-cells were infected with the recombinant adenovirus AdIkappaB((SA)2), which contained a nondegradable mutant form of inhibitory kappaB (IkappaB((SA)2), with S32A and S36A) that locks NF-kappaB in a cytosolic protein complex, preventing its nuclear action. Expression of IkappaB((SA)2) inhibited cytokine-stimulated nuclear translocation and DNA-binding of NF-kappaB. Cytokine-induced gene expression of several NF-kappaB targets, namely inducible nitric oxide synthase, Fas, and manganese superoxide dismutase, was prevented by AdIkappaB((SA)2), as established by reverse transcriptase-polymerase chain reaction, protein blot, and measurement of nitrite in the medium. Finally, beta-cell survival after IL-1beta + IFN-gamma treatment was significantly improved by IkappaB((SA)2) expression, mostly through inhibition of the apoptotic pathway. Based on these findings, we conclude that NF-kappaB activation, under in vitro conditions, has primarily a pro-apoptotic function in beta-cells.

Activation of signal transducer and activator of transcription 3 protects cardiomyocytes from hypoxia/reoxygenation-induced oxidative stress through the upregulation of manganese superoxide dismutase.

BACKGROUND: Mice with cardiac-specific overexpression of signal transducer and activator of transcription 3 (STAT3) are resistant to doxorubicin-induced damage. The STAT3 signal may be involved in the detoxification of reactive oxygen species (ROS). METHODS AND RESULTS: The effects of leukemia inhibitory factor (LIF) or adenovirus-mediated transfection of constitutively activated STAT3 (caSTAT3) on the intracellular ROS formation induced by hypoxia/reoxygenation (H/R) were examined using rat neonatal cardiomyocytes. Either LIF treatment or caSTAT3 significantly suppressed the increase of H/R-induced ROS evaluated by 2',7'-dichlorofluorescin diacetate fluorescence. To assess whether ROS are really involved in H/R-induced cardiomyocyte injury, the amount of creatine phosphokinase in cultured medium was examined. Both LIF treatment and caSTAT3 significantly decreased H/R-induced creatine phosphokinase release. These results indicate that the gp130/STAT3 signal protects H/R-induced cardiomyocyte injury by scavenging ROS generation. To investigate the mechanism of scavenging ROS, the effects of LIF on the induction of antioxidant enzymes were examined. LIF treatment significantly increased the expression of manganese superoxide dismutase (MnSOD) mRNA, whereas the expression of the catalase and glutathione peroxidase genes were unaffected. This induction of MnSOD mRNA expression was completely blocked by adenovirus-mediated transfection of dominant-negative STAT3. Moreover, caSTAT3 augmented MnSOD mRNA and its enzyme activity. In addition, the antisense oligodeoxyribonucleotide to MnSOD significantly inhibited both LIF and caSTAT3-mediated protective effects. CONCLUSIONS: The activation of STAT3 induces a protective effect on H/R-induced cardiomyocyte damage, mainly by inducting MnSOD. The STAT3-mediated signal is proposed as a therapeutical target of ROS-induced cardiomyocyte injury.

Cytokine induction of Fas gene expression in insulin-producing cells requires the transcription factors NF-kappaB and C/EBP.

Fas-mediated cell death may play a role in the autoimmune destruction of pancreatic beta-cells in type 1 diabetes. beta-Cells do not express Fas under physiological conditions, but Fas mRNA and protein are induced in cytokine-exposed mouse and human islets, rendering the beta-cells susceptible to Fas ligand-induced apoptosis. The aim of the present study was to investigate the molecular regulation of Fas by cytokines in rat beta-cells and in insulin-producing RINm5F cells. Fas mRNA expression was increased 15-fold in fluorescence-activated cell sorting-purified rat beta-cells exposed to interleukin (IL)-1beta, whereas gamma-interferon had no effect. Transfection experiments of rat Fas promoter-luciferase reporter constructs into purified rat beta-cells and RINm5F insulinoma cells identified an IL-1beta-responsive region between nucleotides -223 and -54. Inactivation of two adjacent NF-kappaB and C/EBP sites in this region abolished IL-1beta-induced Fas promoter activity in RINm5F cells. Binding of NF-kappaB and C/EBP factors to their respective sites was confirmed by gel shift assays. In cotransfection experiments, NF-kappaB p65 transactivated the Fas promoter. NF-kappaB p50 and C/EBPbeta overexpression had no effect by themselves on the Fas promoter activity, but when cotransfected with p65, each factor inhibited transactivation by p65. These results suggest a critical role for NF-kappaB and C/EBP factors in cytokine-regulation of Fas expression in insulin-producing cells.

Double-stranded ribonucleic acid (RNA) induces beta-cell Fas messenger RNA expression and increases cytokine-induced beta-cell apoptosis.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by progressive destruction of insulin-producing pancreatic beta-cells. Both viral infections and the cytokines interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) have been suggested as potential mediators of beta-cell death in early T1DM. We presently investigated whether the viral replicative intermediate double stranded RNA [here used as synthetic polyinosinic-polycytidylic acid (PIC)] modifies the effects of IL-1beta and IFN-gamma on gene expression and viability of rat pancreatic beta-cells. For this purpose, fluorescence-activated cell sorting-purified rat beta-cells were exposed for 6-16 h (study of gene expression by RT-PCR) or 6-9 days (study of viability by nuclear dyes) to PIC and/or IL-1beta and IFN-gamma. PIC increased the expression of Fas and Mn superoxide dismutase messenger RNAs by 5- to 10-fold. IL-1beta and a combination of PIC and IFN-gamma (but not PIC or IFN-gamma alone) induced expression of inducible nitric oxide (NO) synthase (iNOS) and consequent NO production. Induction of iNOS expression by PIC and IFN-gamma requires nuclear factor-kappaB activation, as suggested by transfection experiments with iNOS promoter-luciferase reporter constructs into primary beta-cells. Combinations of IL-1beta plus IFN-gamma, PIC plus IFN-gamma, or PIC plus IL-1beta induced a 2- to 3-fold increase in the number of apoptotic beta-cells. Blocking of iNOS activity significantly decreased PIC- plus IL-1beta-induced, but not PIC- plus IFN-gamma-induced, apoptosis. In conclusion, PIC alone or in combination with cytokines modifies the expression of several genes in pancreatic beta-cells. Two of these genes, Fas and iNOS, may contribute to beta-cell death. The transcription factor nuclear factor-kappaB is required for PIC-induced iNOS expression. PIC has an additive effect on cytokine-induced beta-cell death by both NO-dependent (in the case of IL-1beta) and NO-independent (in the case of IFN-gamma) mechanisms. These findings suggest that viral intermediates in synergism with local cytokine production may play an important role in beta-cell apoptosis in early T1DM.

beta-cell apoptosis and defense mechanisms: lessons from type 1 diabetes.

Increased evidence suggests that apoptosis is the main mode of beta-cell death in early type 1 diabetes. Cytokines mediate beta-cell apoptosis, and in this article, we discuss some of the cytokine-modified genes that may contribute to beta-cell survival or death. The gene encoding for the inducible form of nitric oxide synthase is induced by interleukin (IL)-1beta or IL-1beta plus gamma-interferon in rodent and human islets, respectively. This leads to nitric oxide (NO) formation, which contributes to a major extent to beta-cell necrosis and to a minor extent to the process of beta-cell apoptosis. The main mode of cell death induced by cytokines in human beta-cells is apoptosis, whereas cytokines lead to both necrosis and apoptosis in rat and mouse beta-cells. It is suggested that the necrotic component in rodent islets is due to NO-induced mitochondrial impairment and consequent decreased ATP production. Human islets, possessing better antioxidant defenses, are able to preserve glucose oxidation and ATP production, and can thus complete the apoptotic program after the death signal delivered by cytokines. We propose that this death signal results from cytokine-induced parallel and/or sequential changes in the expression of multiple proapoptotic and prosurvival genes. The identity of these "gene modules" and of the transcription factors regulating them remains to be established.

Glutathione depletion inhibits IL-1 beta-stimulated nitric oxide production by reducing inducible nitric oxide synthase gene expression.

L-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH synthesis, decreased IL-1 beta-induced nitrite release in rat islets and purified rat beta cells, nitrite formation and iNOS gene promoter activity in insulinoma cells, and iNOS mRNA expression in rat islets. The thiol depletor diethyl maleate (DEM) and an inhibitor of glutathione reductase 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) reduced IL-1 beta-stimulated nitrite release in islets. We conclude that GSH regulates IL-1 beta-induced NO production in islets, purified beta cells and insulinoma cells by modulation of iNOS gene expression.

Outcome and clinical course of 100 patients with adenovirus infection following bone marrow transplantation.

We conducted a retrospective review of the clinical features and outcome of adenovirus infection in 572 consecutive patients transplanted in a single centre over a 10 year period. One hundred patients (17%) had a total of 105 episodes of adenovirus infection diagnosed at a median of 18 days post transplant (range 2-150 days). The incidence was higher in children than adults (21% vs 9%, P < 0.001) and in unrelated donor vs matched sibling donor transplants (26% vs 9%, P < 0.001). Diarrhoea and fever were the most common presenting features. Reflecting these symptoms, the most common site of isolation was the stool. Serotypes 1, 2 and 7 were the most frequently seen (total of 41/68 or 60% of evaluable cases). In six patients (6%) adenovirus infection was the direct cause of death occurring at a median of 72 days post transplant (range 18-365 days). Five of these six patients had pulmonary involvement and four had associated graft-versus-host disease (GVHD). Three further patients were considered to have severe adenoviral disease (total incidence 9%). Isolation of virus from multiple sites correlated with a poor outcome (P < 0.001). Comorbid viral infection was common in this group with 50% of all patients having other viruses isolated (predominantly polyoma virus and cytomegalovirus). We conclude that adenovirus is commonly isolated after bone marrow transplant and is a cause of significant morbidity but was a rare cause of mortality (6/572 = 1%) in our patient group as a whole. The relative infrequency of severe infection will make it difficult for the transplant physician to decide which patients should receive experimental antiviral drugs such as ribavirin and cidofovir or immunomodulatory therapy with donor white cell infusions.

NF-kappaB is required for cytokine-induced manganese superoxide dismutase expression in insulin-producing cells.

Reactive oxygen species play an important role in the cytotoxic effect of inflammatory cytokines on pancreatic beta-cells in type 1 diabetes mellitus. The antioxidant enzyme manganese superoxide dismutase (MnSOD) is part of the cellular defenses against these deleterious radicals. MnSOD gene expression is induced by cytokines in insulin-producing cells, but the transcriptional regulation of MnSOD expression in these cells is not well understood. In this report, we investigated the transcriptional regulation by cytokines of the rat MnSOD gene in insulin-producing cells. By transient transfections with promoter-luciferase reporter constructs, we identified two interleukin (IL)-1beta-responsive elements, conferring each an additive 3-fold IL-1beta-induced transcriptional activity. The first is located in the promoter region, whereas the second is located in the second intron of the MnSOD gene. Interestingly, the intronic element is required for interferon-gamma-induced potentiation. Site-directed mutagenesis and band-shift assays showed that an NF-kappaB binding site in each region is necessary, but not sufficient, for transcriptional induction by IL-1beta. Our results suggest that NF-kappaB may cooperate with CCAAT/enhancer-binding protein factors in the promoter region and with octamer and Ets factors in the intronic region.

Apoptosis induced by growth factor withdrawal in fibroblasts overproducing fructose 2,6-bisphosphate.

Fructose 2,6-bisphosphate is a potent endogenous stimulator of glycolysis. A high aerobic glycolytic rate often correlates with increased cell proliferation. To investigate this relationship, we have produced clonal cell lines of Rat-1 fibroblasts that stably express transgenes coding for 6-phosphofructo-2-kinase, which catalyzes the synthesis of fructose 2,6-bisphosphate, or for fructose 2,6-bisphosphatase, which catalyzes its degradation. While serum deprivation in culture reduced the growth rate of control cells, it caused apoptosis in cells overproducing fructose 2,6-bisphosphate. Apoptosis was inhibited by 5-amino-4-imidazolecarboxamide riboside, suggesting that 5'-AMP-activated protein kinase interferes with this phenomenon.

Regulation by cytokines of the inducible nitric oxide synthase promoter in insulin-producing cells.

Cytokines could contribute to beta-cell damage in Type I diabetes mellitus. The radical nitric oxide, generated by the inducible form of nitric oxide synthase (iNOS), is a potential mediator of cytokine-induced beta-cell dysfunction. In rat pancreatic islets and insulin-producing cell lines, interleukin-1beta (IL-1beta) induces expression of iNOS mRNA and increases NO production, an effect potentiated by interferon-gamma (IFN-gamma). In human islet cells both IL-1beta and IFN-gamma are required for iNOS expression. We have shown previously that both the transcription factors nuclear factor-kappaB (NF-kappaB) and interferon regulatory factor-1 (IRF-1) are activated by cytokines in rodent and human islets but there is no direct information on the regulation of the iNOS promoter in insulin-producing cells. We presently investigated the effects of cytokines on iNOS transcriptional regulation in both rat insulin-producing RINm5F cells and in primary FACS-purified rat beta cells. Transient transfection experiments with the 1.5-kb rat promoter region and 5' deletants of it showed that a distal region extending up to -1002 bp, and containing a distal and a proximal nuclear factor-kappaB (NF-kappaB) binding site, a gamma-interferon activated site (GAS) and two adjacent IFN-stimulated response elements (ISRE), is required for IL-1beta induction and IFN-gamma potentiation of iNOS activation. Site-mutation analysis showed that both the distal and proximal NF-kappaB and GAS are necessary for IL-1beta-induced iNOS expression in RINm5F cells. In these cells IFN-gamma potentiation is mostly mediated by GAS and ISRE, suggesting a role for the IFN-gamma-induced transcription factors Stat1alpha (which binds GAS) and IRF-1 (which binds ISRE), which may cooperate with NF-kappaB induced by IL-1beta for iNOS activation. In primary beta cells both NF-kappaB binding sites are required for IL-1beta-induced iNOS promoter activation. In these cells IFN-gamma neither increased IL-1beta-induced iNOS promoter activity nor iNOS mRNA expression but it induced a twofold increase in NO production. The present results unveiled the nature of the promoter binding sites necessary for iNOS expression in rodent beta cells. This information could be relevant for the development of new strategies aimed at preventing cytokine-induced iNOS expression and consequent beta-cell damage.

E2F-dependent mitogenic stimulation of the splicing of transcripts from an S phase-regulated gene.

There is one class of genes whose expression increases at the G1/S transition of the cell cycle. One of these genes codes for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2), an enzyme that controls glycolysis. The cell-cycle regulation of the PFK-2 gene depends on a binding site for the transcription factor E2F located at the 5'end of the first exon and involves not only a transcriptional, but also a post-transcriptional, mechanism. We have investigated this mechanism by studying in Rat-1 fibroblasts mature and immature mRNAs from the endogenous PFK-2 gene and from stably expressed transgenes containing PFK-2 gene regions. An increase in precursor mRNA half-life and processing took place at the G1/S transition. Transgenes with a mutated E2F binding site or with mutated splice sites lost the regulation by serum, indicating that both an intact E2F binding site and an efficient splicing reaction are necessary for proper mitogenic stimulation. In quiescent cells, the transgene lacking the E2F binding site was more efficiently spliced than the wild-type construct. These results indicate that, in the wild-type gene, precursor mRNA splicing is blocked in G0and that this block requires the E2F binding site. The data provide evidence for a coupling between stimulation of promoter activity and increased mRNA splicing in the mitogenic regulation of S phase-regulated genes.

An E2F-dependent late-serum-response promoter in a gene that controls glycolysis.

The F-type mRNA of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is found in proliferating, but not in quiescent, cells. This bifunctional enzyme catalyses the synthesis and degradation of fructose-2,6-bisphosphate, a potent stimulator of glycolysis. F-type mRNA concentration decreased upon differentiation of rat rhabdomyosarcoma cells; it increased in Rat-1 fibroblasts stimulated to proliferate by serum, by epidermal growth factor, or by the v-src oncogene product. This increase resulted, at least in part, from a stimulation of F promoter activity. The stimulation occurred at the G1/S transition of the cell cycle. It depended on a binding site for the oncogenic transcription factor E2F located in the first exon of F-type mRNA. This effect was inhibited by agents that increase cAMP concentration. The data provide the first evidence that a gene involved in the control of glycolysis can be regulated by a late-serum-response promoter in an E2F-dependent way.

Characterization of the promoter of the gene encoding human tissue inhibitor of metalloproteinases-2 (TIMP-2).

Tissue inhibitors of metalloproteinases (TIMPs) are multifunctional proteins that control the proteolytic activity of matrix metalloproteinases (MMPs). We report here the cloning and characterization of a 2.5-kb genomic fragment of the human timp-2 gene that includes 519 bp of the 5' flanking region, the first coding exon (432-bp) and part of the first intron. The 5' flanking region has several features of housekeeping genes. It has a high G-C content and is included in a typical CpG island. It also contains a TATA-like element (AATAAAA) located 23 to 37-bp upstream from a cluster of transcription start points (tsp), several Sp1 and one AP-2 motifs, and an AP-1 consensus sequence located at position -590 to -583 from the start codon. When inserted upstream from a promoterless luciferase-encoding gene, a 715-bp fragment of this 5'-flanking sequence behaved as a promoter in transiently transfected NIH3T3 and Rat-1 fibroblasts. The effect of deletions of the promoter suggested the presence of a negative control element located between positions -661 and -575. This element includes the AP-1 consensus sequence. However, treatment with phorbol did not change activity in transfected cells and did not change the timp-2 mRNA content of human HT1080 fibrosarcoma cells. A comparison with the promoter of murine timp-1 revealed several differences consistent with the fact that timp-1 and timp-2 are differentially regulated.

Characterization of a hepatoma mRNA transcribed from a third promoter of a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-encoding gene and controlled by ets oncogene-related products.

6-Phosphofructo-2-kinase (EC 2.7.1.105)/fructose-2,6-bis-phosphatase (EC 3.1.3.46) catalyzes the synthesis and degradation of fructose 2,6-bisphosphate, a ubiquitous stimulator of glycolysis. The liver (L-type) and muscle (M-type) mRNAs for this bifunctional enzyme arise from distinct promoters of the same gene. We have now characterized in rat hepatoma FTO2B cells another mRNA, which is transcribed from a third promoter of that gene. This F-type mRNA is present in fetal rat liver and muscle, in rat placenta, and in several established rat cell lines. The F promoter contains no TATA box but contains several binding sites for Sp1 and for members of the ets oncogene family. Transfection of FTO2B cells with constructs containing the intact or mutagenized F promoter showed that its activity depends mainly on one of these sites. This site bound a heteromeric FTO2B cell protein indistinguishable from the ets-related GA binding protein alpha/ankyrin-repeats GA binding protein beta transcription factor.

Characterization of an enhancer upstream from the muscle-type promoter of a gene encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The muscle-type isozyme of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is encoded by a mRNA transcribed from the M promoter of a 55-kb gene, which also produces the liver-type isozyme from an alternative promoter. By transient transfection and in vitro protein-DNA binding assays we have delineated, within 4.7 kb of 5' flanking sequence, the M promoter proper and an enhancer located between -1615 and -1809. This enhancer stimulated up to 12-fold the activity of the promoter in the context of an intact 5' flanking sequence and close to 900-fold the activity of the minimal (+41 to -40) M promoter cloned directly downstream from it. A functional dissection of the enhancer by site-directed mutagenesis and use of oligonucleotides suggested that its activity involves the cooperative effect of six binding sites for trans-acting factors clustered within 150 bp. These sites contain either an EF-1A/E4TF1 motif (also known to bind the ets oncogene product) or a Sp1 motif, or both. The activity of the enhancer could be demonstrated in L6 myoblasts and myocytes and in FTO2B hepatoma cells. When left within the intact 5' flanking sequence, however, enhancer activity was inhibited upon differentiation of myoblasts into myocytes.

Human and rat chromosomal localization of two genes for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by analysis of somatic cell hybrids and in situ hybridization.

Two genes encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were localized in human and rat chromosomes. PFKFB1 (previously PFRX), which encodes the liver and muscle isozymes, was assigned to Xq22-q31 in the rat and to Xq27-q28 in the human by in situ hybridization using probes generated by the polymerase chain reaction. PFKFB2, which encodes the heart isozyme of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was assigned to chromosome 13 in the rat and to chromosome 1 in the human by hybridization of DNA from somatic cell hybrids. By in situ hybridization, this gene was localized to the regions 13q24-25 in the rat and 1q31 in the human.