Bleomycin upregulates expression of gamma-glutamylcysteine synthetase in pulmonary artery endothelial cells.
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The chemotherapeutic agent bleomycin induces pulmonary fibrosis through the generation of reactive oxygen species (ROS), which are thought to contribute to cellular damage and pulmonary injury. We hypothesized that bleomycin activates oxidative stress response pathways and regulates cellular glutathione (GSH). Bovine pulmonary artery endothelial cells exposed to bleomycin exhibit growth arrest and increased cellular GSH content. gamma-Glutamylcysteine synthetase (gamma-GCS) controls the key regulatory step in GSH synthesis, and Northern blots indicate that the gamma-GCS catalytic subunit [gamma-GCS heavy chain (gamma-GCS(h))] is upregulated by bleomycin within 3 h. The promoter for human gamma-GCS(h) contains consensus sites for nuclear factor-kappaB (NF-kappaB) and the antioxidant response element (ARE), both of which are activated in response to oxidative stress. Electrophoretic mobility shift assays show that bleomycin activates the transcription factor NF-kappaB as well as the ARE-binding factors Nrf-1 and -2. Nrf-1 and -2 activation by bleomycin is inhibited by the ROS quenching agent N-acetylcysteine (NAC), but not by U-0126, a MEK1/2 inhibitor that blocks bleomycin-induced MAPK activation. In contrast, NF-kappaB activation by bleomycin is inhibited by U-0126, but not by NAC. NAC and U-0126 both inhibit bleomycin-induced upregulation of gamma-GCS expression. These data suggest that bleomycin can activate oxidative stress response pathways and upregulate cellular GSH. (+info)
Mutation of a nuclear respiratory factor 2 binding site in the 5' untranslated region of the ADSL gene in three patients with adenylosuccinate lyase deficiency.
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Adenylosuccinate lyase (ADSL; also called "adenylosuccinase") catalyzes two steps in the synthesis of purine nucleotides: (1) the conversion of succinylaminoimidazolecarboxamide ribotide into aminoimidazolecarboxamide ribotide and (2) the conversion of adenylosuccinate into adenosine monophosphate. ADSL deficiency, a recessively inherited disorder, causes variable-but most often severe-mental retardation, frequently accompanied by epilepsy and/or autism. It is characterized by the accumulation, in body fluids, of succinylaminoimidazolecarboxamide riboside and succinyladenosine, the dephosphorylated derivatives of the two substrates of the enzyme. Analysis of the ADSL gene of three unrelated patients with ADSL deficiency, in whom one of the ADSL alleles displayed a normal coding sequence, revealed a -49T-->C mutation in the 5' untranslated region of this allele. Measurements of the amount of mRNA transcribed from the latter allele showed that it was reduced to approximately 33% of that transcribed from the alleles mutated in their coding sequence. Further investigations showed that the -49T-->C mutation provokes a reduction to 25% of wild-type control of promoter function, as evaluated by luciferase activity and mRNA level in transfection experiments. The mutation also affects the binding of nuclear respiratory factor 2 (NRF-2), a known activator of transcription, as assessed by gel-shift studies. Our findings indicate that a mutation of a regulatory region of the ADSL gene might be an unusually frequent cause of ADSL deficiency, and they suggest a role for NRF-2 in the gene regulation of the purine biosynthetic pathway. (+info)
Mitochondrial transcription factor A and its downstream targets are up-regulated in a rat hepatoma.
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Mitochondrial transcription factor A is a key regulator involved in mitochondrial DNA transcription and replication. In a poorly differentiated rat hepatoma, Morris hepatoma 3924A, the mRNA and protein levels of this factor were elevated about 10- and 11-fold, respectively, relative to the host liver. The mRNA levels for the hepatoma cytochrome c oxidase I, II, and NADH dehydrogenase 5, 6, the downstream targets of Tfam, were augmented 10-, 8-, 5-, and 3-fold, respectively. Interestingly, Tfam was also found in the hepatoma nucleus. The mRNA levels for nuclear respiratory factor 1 and 2 (NRF-1 and -2), the proteins that are known to interact with specific regulatory elements on human TFAM promoter, were 5- and 3-fold higher, respectively, in the hepatoma relative to the host liver. Unlike the human promoter, the rat Tfam promoter did not form a specific complex with the NRF-1 in the liver or hepatoma nuclear extracts, which is consistent with the absence of an NRF-1 consensus sequence in the proximal rat promoter. A single specific complex formed between the rat promoter and the NRF-2 protein was comparable in the two extracts. The DNA binding activity of Sp1 in the hepatoma nuclear extract was 4-fold greater than that in the liver extract. In vivo genomic footprinting showed occupancy of NRF-2 and Sp1 consensus sites on the promoter of rat Tfam gene. Tfam was also up-regulated in other hepatoma cells. Together, these results show up-regulation of Tfam in some tumors, particularly the liver tumors. Further, the relatively high level of Sp1 binding to the promoter in the hepatoma could play a major role in the up-regulation of Tfam in these tumor cells. (+info)
Isolation and characterization of chicken GA-binding protein.
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We have purified and characterized chicken liver nuclear proteins that bind to Ets binding sites (EBSs) of ribosomal protein (r-protein) gene promoters. We employed supershift assays and antibodies to mouse GA binding protein (GABP), to show that the proteins were similar to alpha and beta subunits of GABP. Western blot analysis identified 54- and 38-kDa proteins as the alpha type, and a 46-kDa protein as the beta type. When compared with nuclear extracts (NEs) of other species, we observed that the 38-kDa protein was unique to chicken, and appears to be derived from the 54-kDa protein. The 54- and 46-kDa proteins were highly expressed in chicken tissues and were major components through higher animals, indicating that both proteins have a conserved role. (+info)
Adaptations of skeletal muscle to exercise: rapid increase in the transcriptional coactivator PGC-1.
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Endurance exercise induces increases in mitochondria and the GLUT4 isoform of the glucose transporter in muscle. Although little is known about the mechanisms underlying these adaptations, new information has accumulated regarding how mitochondrial biogenesis and GLUT4 expression are regulated. This includes the findings that the transcriptional coactivator PGC-1 promotes mitochondrial biogenesis and that NRF-1 and NRF-2 act as transcriptional activators of genes encoding mitochondrial enzymes. We tested the hypothesis that increases in PGC-1, NRF-1, and NRF-2 are involved in the initial adaptive response of muscle to exercise. Five daily bouts of swimming induced increases in mitochondrial enzymes and GLUT4 in skeletal muscle in rats. One exercise bout resulted in approximately twofold increases in full-length muscle PGC-1 mRNA and PGC-1 protein, which were evident 18 h after exercise. A smaller form of PGC-1 increased after exercise. The exercise induced increases in muscle NRF-1 and NRF-2 that were evident 12 to 18 h after one exercise bout. These findings suggest that increases in PGC-1, NRF-1, and NRF-2 represent key regulatory components of the stimulation of mitochondrial biogenesis by exercise and that PGC-1 mediates the coordinated increases in GLUT4 and mitochondria. (+info)
GA-binding protein (GABP) and Sp1 are required, along with retinoid receptors, to mediate retinoic acid responsiveness of CD18 (beta 2 leukocyte integrin): a novel mechanism of transcriptional regulation in myeloid cells.
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CD18 (beta(2) leukocyte integrin) is transcriptionally regulated in myeloid cells, but the mechanisms that increase its expression in response to retinoic acid (RA) have not been defined. The CD18 promoter was activated by RA treatment in stably transfected U937 myeloid cells. We identified a retinoic acid response element (RARE) that lies nearly 900 nucleotides upstream of the CD18 transcriptional start site that was bound by the RA receptors, retinoic acid receptor (RAR) and retinoic X receptor (RXR). This RARE accounted for one half of the RA responsiveness of CD18. However, unexpectedly, one half of the dynamic response to RA was mediated by the 96-nucleotide CD18 minimal promoter, which lacks a recognizable RARE. Binding sites for the ets transcription factor, GA-binding protein (GABP), and Sp1 were required for full RA responsiveness of both the CD18 minimal promoter and the full-length promoter. The ets sites conferred RA responsiveness on an otherwise unresponsive heterologous promoter, and RA responsiveness was directly related to the number of ets sites. The transcriptional coactivator p300/CBP physically interacted with GABP in vivo, and p300 increased the responsiveness of the CD18 promoter to RA. These studies demonstrate a novel role for non-RAR transcription factors in mediating RA activation in myeloid cells. They support the concept that transcription factors other than RARs are required for RA-activated gene expression. We hypothesize that a multiprotein complex--an enhanceosome--that includes GABP, other transcription factors, and coactivators, dynamically regulates CD18 expression in myeloid cells. (+info)
Purification and mass spectrometric identification of GA-binding protein (GABP) as the functional pituitary Ets factor binding to the basal transcription element of the prolactin promoter.
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The Ets-binding site within the basal transcription element (BTE) of the rat prolactin (rPRL) promoter is critical for both basal and growth factor-regulated rPRL gene expression. Here we report the purification and identification of the factor that binds to the BTE. This factor was purified from GH3 pituitary nuclear extracts using ammonium sulfate fractionation, heparin-Sepharose and Mono Q chromatography, and BTE-affinity magnetic beads. We purified two proteins of 57 and 47 kDa and identified the 57-kDa protein by mass spectrometry as the Ets factor GABPalpha. Western blot analysis identified the 47-kDa protein as GABPbeta1. Co-transfection of dominant-negative GABPbeta1 blocks prolactin promoter basal activity by 85-88% in GH3 cells in the presence or absence of FGF-4. Additionally, expression of wild-type GABPalpha/beta1 selectively activates a minimal BTE promoter 24-28-fold in GH3 cells, and this activation is dependent on the Ets-binding site. Finally, small interfering RNA depletion of GABP in GH3 cells results in the loss of prolactin protein. Thus, we have identified GABPalpha/GABPbeta1 as a critical and functionally relevant Ets factor that regulates rPRL promoter activity via the BTE site. (+info)
Raising Ca2+ in L6 myotubes mimics effects of exercise on mitochondrial biogenesis in muscle.
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Skeletal muscle adapts to endurance exercise with an increase in mitochondria. Muscle contractions generate numerous potential signals. To determine which of these stimulates mitochondrial biogenesis, we are using L6 myotubes. Using this model we have found that raising cytosolic Ca2+ induces an increase in mitochondria. In this study, we tested the hypothesis that raising cytosolic Ca2+ in L6 myotubes induces increased expression of PGC-1, NRF-1, NRF-2, and mtTFA, factors that have been implicated in mitochondrial biogenesis and in the adaptation of muscle to exercise. Raising cytosolic Ca2+ by exposing L6 myotubes to caffeine for 5 h induced significant increases in PGC-1 and mtTFA protein expression and in NRF-1 and NRF-2 binding to DNA. These adaptations were prevented by dantrolene, which blocks Ca2+ release from the SR. Exposure of L6 myotubes to caffeine for 5 h per day for 5 days induced significant increases in mitochondrial marker enzyme proteins. Our results show that the adaptive response of L6 myotubes to an increase in cytosolic Ca2+ mimics the stimulation of mitochondrial biogenesis by exercise. They support the hypothesis that an increase in cytosolic Ca2+ is one of the signals that mediate increased mitochondrial biogenesis in muscle. (+info)