The mechanism by which disruption of reading frame can influence pre-messenger RNA (pre-mRNA) processing is poorly understood. We assessed the role of factors essential for nonsense-mediated mRNA ...decay (NMD) in nonsense-mediated altered splicing (NAS) with the use of RNA interference (RNAi) in mammalian cells. Inhibition of rent1/hUpf1 expression abrogated both NMD and NAS of nonsense T cell receptor β transcripts. In contrast, inhibition of rent2/hUpf2 expression did not disrupt NAS despite achieving comparable stabilization of nonsense transcripts. We also demonstrate that NAS and NMD are genetically separable functions of rent1/hUpf1. Additionally, rent1/hUpf1 enters the nucleus where it may directly influence early events in mRNA biogenesis. This provides compelling evidence that NAS relies on a component of the nonsense surveillance machinery but is not an indirect consequence of NMD.
Loeys-Dietz syndrome (LDS) is a connective tissue disorder that is characterized by a high risk for aneurysm and dissection throughout the arterial tree and phenotypically resembles Marfan syndrome. ...LDS is caused by heterozygous missense mutations in either TGF-β receptor gene (TGFBR1 or TGFBR2), which are predicted to result in diminished TGF-β signaling; however, aortic surgical samples from patients show evidence of paradoxically increased TGF-β signaling. We generated 2 knockin mouse strains with LDS mutations in either Tgfbr1 or Tgfbr2 and a transgenic mouse overexpressing mutant Tgfbr2. Knockin and transgenic mice, but not haploinsufficient animals, recapitulated the LDS phenotype. While heterozygous mutant cells had diminished signaling in response to exogenous TGF-β in vitro, they maintained normal levels of Smad2 phosphorylation under steady-state culture conditions, suggesting a chronic compensation. Analysis of TGF-β signaling in the aortic wall in vivo revealed progressive upregulation of Smad2 phosphorylation and TGF-β target gene output, which paralleled worsening of aneurysm pathology and coincided with upregulation of TGF-β1 ligand expression. Importantly, suppression of Smad2 phosphorylation and TGF-β1 expression correlated with the therapeutic efficacy of the angiotensin II type 1 receptor antagonist losartan. Together, these data suggest that increased TGF-β signaling contributes to postnatal aneurysm progression in LDS.
The Fibroblast Growth Factor (FGF) signaling pathway is reported to stimulate glioblastoma (GBM) growth. In this work we evaluated the effect of FGF2, FGF receptor (FGFR), and small molecule ...inhibition on GBM cells grown in traditional media, or cultured directly in stem-cell media. These lines each expressed the FGFR1, FGFR3 and FGFR4 receptors. Addition of FGF2 ligand showed significant growth stimulation in 8 of 10 cell lines. Disruption of FGF signaling by a neutralizing FGF2 monoclonal antibody and FGFR1 suppression by RNA interference both partially inhibited cell proliferation. Growth inhibition was temporally correlated with a reduction in MAPK signaling. A receptor tyrosine kinase inhibitor with known FGFR/VEGFR activity, PD173074, showed reproducible growth inhibition. Possible mechanisms of growth suppression by PD173074 were implicated by reduced phosphorylation of AKT and MAPK, known oncogenic signal transducers. Subsequent reduction in the cyclin D1, cyclin D2 and CDK4 cell cycle regulators was also observed. Our results indicate that FGF signaling pathway inhibition as a monotherapy will slow, but not arrest growth of glioblastoma cells.
Loeys-Dietz syndrome (LDS) is a connective tissue disorder that is characterized by a high risk for aneurysm and dissection throughout the arterial tree and phenotypically resembles Marfan syndrome. ...LDS is caused by heterozygous missense mutations in either TGF-betareceptor gene (TGFBR1 or TGFBR2), which are predicted to result in diminished TGF-beta signaling; however, aortic surgical samples from patients show evidence of paradoxically increased TGF-beta signaling. We generated 2 knockin mouse strains with LDS mutations in either Tgfbr1 or Tgfbr2 and a transgenic mouse overexpressing mutant Tgfbr2. Knockin and transgenic mice, but not haploinsufficient animals, recapitulated the LDS phenotype. While heterozygous mutant cells had diminished signaling in response to exogenous TGF-beta in vitro, they maintained normal levels of Smad2 phosphorylation under steady-state culture conditions, suggesting a chronic compensation. Analysis of TGF-beta signaling in the aortic wall in vivo revealed progressive upregulation of Smad2 phosphorylation and TGF-beta target gene out-put, which paralleled worsening of aneurysm pathology and coincided with upregulation of TGF-beta 1 ligand expression. Importantly, suppression of Smad2 phosphorylation and TGF-beta 1 expression correlated with the therapeutic efficacy of the angiotensin II type 1 receptor antagonist losartan. Together, these data suggest that increased TGF-beta signaling contributes to postnatal aneurysm progression in LDS.
Reactive oxygen species have been shown to generate mutagenic lesions in DNA. One of the most abundant lesions in both nuclear and mitochondrial DNA is 7,8-dihydro-8-oxoguanine (8-oxoG). We report ...here the partial purification and characterization of a mitochondrial oxidative damage endonuclease (mtODE) from rat liver that recognizes and incises at 8-oxoG and abasic sites in duplex DNA. Rat liver mitochondria were purified by differential and Percoll gradient centrifugation, and mtODE was extracted from Triton X-100-solubilized mitochondria. Incision activity was measured using a radiolabeled double-stranded DNA oligonucleotide containing a unique 8-oxoG, and reaction products were separated by polyacrylamide gel electrophoresis. Gel filtration chromatography predicts mtODE's molecular mass to be between 25 and 30 kDa. mtODE has a monovalent cation optimum between 50 and 100 mm KCl and a pH optimum between 7.5 and 8. mtODE does not require any co-factors and is active in the presence of 5 mm EDTA. It is specific for 8-oxoG and preferentially incises at 8-oxoG:C base pairs. mtODE is a putative 8-oxoG glycosylase/lyase enzyme, because it can be covalently linked to the 8-oxoG oligonucleotide by sodium borohydride reduction. Comparison of mtODE's activity with other known 8-oxoG glycosylases/lyases and mitochondrial enzymes reveals that this may be a novel protein.
CCAAT/enhancer-binding proteins (C/EBPs) comprise a homologous group of transcriptional regulators that control liver and
fat differentiation and are involved in regulating the expression of acute ...phase reactants during the host response to inflammation.
GADD153, a unique member of the C/EBP family, has been proposed to act as a dominant negative inhibitor of other C/EBPs, but
little is known about its expression in liver or its role in the processes described above. We have examined its expression
during the acute phase response (APR) and have shown that like C/EBP beta and C/EBP delta, GADD153 mRNA is markedly induced
in livers of rats treated with lipopolysaccharide to initiate the APR. Interestingly, its induction is temporally delayed
relative to that of C/EBP beta and C/EBP delta but is similar to that of acute phase reactants shown to be regulated by C/EBPs.
Footprint analysis of the GADD153 promoter showed binding of proteins in liver extracts of both untreated and lipopolysaccharide-injected
rats to a putative C/EBP regulatory site. Gel shift analysis showed that although present constitutively, binding activity
was increased in extracts from lipopolysaccharide-treated animals. Both C/EBP alpha and C/EBP beta were shown to contribute
to the binding activity with the contribution by C/EBP beta increasing during the APR. Support for the functional role of
this C/EBP-binding site and its interaction with C/EBPs in regulating GADD153 expression was obtained with cultured HepG2
hepatoma cells in which overexpression of C/EBP beta was found to transactivate expression of a plasmid containing the GADD153
promoter linked to a reporter gene. These findings suggest that the GADD153 gene is itself regulated by C/EBPs during the
host response to inflammation and that GADD153 is likely to contribute to the regulation of other genes whose expression is
altered during the APR.
Transcriptional activation of c-fos in response to both serum stimulation and DNA damage requires the serum response element. The inability of in vitro aged or senescent fibroblasts to proliferate in ...response to serum has been shown to be associated with repressed c-fos expression and reduced AP-1 binding activity. In contrast, we have observed similar levels of c-fos mRNA and protein expression in young (early passage) and old (late passage) cells following their treatment with ultraviolet (UV) irradiation or methyl methanesulfonate (MMS). Thus, the early events in the signal transduction pathway leading to transcriptional activation of c-fos following DNA damage are distinct from those mediating the gene's expression in response to mitogenic stimulation. Despite normal levels of c-fos expression, we observed a reduced level of AP-1 binding activity in old cells relative to young cells treated with UV irradiation or MMS. Reduced AP-1 binding activity is associated with reduced expression of the AP-1-dependent gene, collagenase, in old cells treated with DNA damaging agents. Since other DNA damage-inducible genes also contain an AP-1 regulatory element presumed to play a role in their expression, reduced AP-1 binding activity is likely to have a major impact on the old cell's ability to respond appropriately to DNA damage.
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