FOXO3a-dependent up-regulation of Mxi1-0 promotes hypoxia-induced apoptosis in endothelial cells

Endothelial cell apoptosis induced by hypoxia is implicated in the pathogenesis of vascular diseases. However, the underlying mechanism is not clearly elucidated. In this study, we found that hypoxia increased Mxi1-0 expression, and the Mxi1-0 siRNA could inhibit caspase-8 activation and apoptosis in HUVECs induced by hypoxia. In addition, hypoxia induced FOXO3 activation, while Mxi1-0 expression and apoptosis were inhibited by transfection with FOXO3 siRNA. Using ChIP assay, we confirmed that FOXO3a binds to the Mxi1-0 promoter region. Furthermore, hypoxia treatment leads to remarkable production of reactive oxygen species (ROS), while ROS scavenger N-acetyl-L-cysteine (NAC) inhibits hypoxia-induced ROS production, apoptosis and FOXO3a-mediated Mxi1-0 up-regulation. Finally, we found that the HIF-1α siRNA inhibited hypoxia-induced HIF-1α expression and ROS production, as well as FOXO3a/Mxi1-0 activation and apoptosis in HUVECs. Taken together, this study identifies a HIF-1α/FOXO3a/Mxi1-0/caspase-8 signaling pathway in hypoxia-induced endothelial cell apoptosis. These data also indicate that HIF-1α-dependent ROS production is required for FOXO3a-mediated Mxi1-0 up-regulation and apoptosis in hypoxic endothelial cells.     

FOXO 1a and FOXO 3a gene polymorphisms in association with metabolic syndrome

The prevalence and magnitude of childhood and adult obesity and diabetes are increasing dramatically. FOXO 1a and FOXO 3a will be evaluated in this study, in an effort to identify genetic polymorphisms in potential candidate genes that may be associated with body mass index (BMI), and metabolic syndrome (MS). Also to assess whether there is a relation between insulin sensitivity, and genotype, we will test the relation between fasting insulin, glucose, insulin resistance, insulin secretion and genotype.A total number of 248 presenting normal, overweight and obese individuals were recruited; 100 children and 148 adults of both sexes. They were divided by body mass index as follows, normal, overweight and obese. Lipid profile, fasting glucose and insulin HOMA-IR and HOMA-β index and RT-PCR for FOXO 1a and FOXO 3a were performed.An association was found among the studied group (children and adults) as regards foxo3a gene polymorphism and HOMA IR, HOMA B index and T-cholesterol (P = 0.022, 0.011 and 0.028, respectively), while there was only an association between LDL-C and foxo1a gene polymorphism among the studied group of children and adults (P = 0.023).In this study we demonstrated that FOXO3a mutant is correlated with HOMA-IR (marker of insulin resistance), HOMA-B index (marker of insulin secretion) and total cholesterol while as regards FOXO1a there was only an association between LDL cholesterol and mutant type of FOXO1a.     

Transcriptional repression of human cad gene by hypoxia inducible factor-1alpha

De novo biosynthesis of pyrimidine nucleotides provides essential precursors for DNA synthesis and cell proliferation. The first three steps of de novo pyrimidine biosynthesis are catalyzed by a multifunctional enzyme known as CAD (carbamoyl phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase). In this work, a decrease in CAD expression is detected in numerous cell lines and primary culture human stromal cells incubated under hypoxia or desferrioxamine (DFO)-induced HIF-1alpha accumulation. A putative hypoxia response element (HRE) binding matrix is identified by analyzing human cad-gene promoter using a bioinformatic approach. Promoter activity assays, using constructs harboring the cad promoter (-710/+122) and the -67/HRE fragment (25-bases), respectively, demonstrate the suppression of reporter-gene expression under hypoxia. Suppression of cad-promoter activity is substantiated by forced expression of wild-type HIF-1alpha but abolished by overexpression of dominant-negative HIF-1alpha. A chromatin immunoprecipitation assay provides further evidence that HIF-1alpha binds to the cad promoter in vivo. These data demonstrate that the cad-gene expression is repressed by HIF-1alpha, which represents a functional link between hypoxia and cell-cycle arrest.     

Induction of OGG1 gene expression by HIV-1 Tat

To identify the cellular gene target for Tat, we performed gene expression profile analysis and found that Tat up-regulates the expression of the OGG1 (8-oxoguanine-DNA glycosylase-1) gene, which encodes an enzyme responsible for repairing the oxidatively damaged guanosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). We observed that Tat induced OGG1 gene expression by enhancing its promoter activity without changing its mRNA stability. We found that the upstream AP-4 site within the OGG1 promoter is responsible and that Tat interacted with AP-4 and removed AP-4 from the OGG1 promoter by in vivo chromatin immunoprecipitation assay. Thus, Tat appears to activate OGG1 expression by sequestrating AP-4. Interestingly, although Tat induces oxidative stress known to generate 8-oxo-dG, which causes the G:C to T:A transversion, we observed that the amount of 8-oxo-dG was reduced by Tat. When OGG1 was knocked down by small interfering RNA, Tat increased the amount of 8-oxo-dG, thus confirming the role of OGG1 in preventing the formation of 8-oxo-dG. These findings collectively indicate the possibility that Tat may play a role in maintenance of the genetic integrity of the proviral and host cellular genomes by up-regulating OGG1 as a feed-forward mechanism.