E1B 19-kDa Protein-interacting Protein Research Articles

Increased activation of the hypoxia-inducible factor pathway in varicose veins

Venous hypoxia has been postulated to contribute to varicose vein (VV) formation. Direct measurements of vein wall oxygen tension have previously demonstrated that the average minimum oxygen tensions were significantly lower in VVs compared with non-varicose veins (NVVs). Hypoxia-inducible factors (HIFs) are nuclear transcriptional factors that regulate the expression of several genes of oxygen homeostasis. This study aimed to investigate if hypoxia was associated with VVs by assessing the expression of HIF-1α, HIF-2α, HIF target genes, and upstream HIF regulatory enzymes in VVs and NVVs, and their regulation by hypoxia. VVs and NVVs were surgically retrieved and immediately snap-frozen or used for organ culture preparation. The relative expression of HIF-1α, HIF-2α, HIF target genes, and HIF regulatory enzymes in VVs and NVVs was analyzed with quantitative polymerase chain reaction (Q-PCR) and Western blot. VV and NVV organ ex vivo cultures were exposed to 16 hours of normoxia, hypoxia (oxygen tension 1%), or the hypoxia mimetic dimethyloxallyl glycine (DMOG) 1 mM in normoxia. The vein organ cultures were then analyzed for HIF-1α, HIF-2α, and their target gene expression with Q-PCR and Western blot. HIF-1α and HIF-2α mRNA were significantly upregulated in VVs compared with NVVs (89.8 ± 18.6 vs 10.4 ± 7.2 and 384.9 ± 209.4 vs 8.1 ± 4.2, respectively). HIF target gene mRNA expression was also significantly elevated in VVs compared with NVVs, namely glucose transporter-1 (GLUT-1; 8.7 ± 2.1 vs 1.0 ± 0.3), carbonic anhydrase-9 (CA9; 8.5 ± 2.1 vs 2.8 ± 1.2), vascular endothelial growth factor (VEGF; 7.5 ± 2.1 vs 0.9 ± 0.2), and BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP-3; 4.5 ± 0.7 vs 1.4 ± 0.3). The upregulation of HIF-1α, HIF-2α, and HIF target genes in VVs was also reflected at protein level. Of the HIF regulatory enzymes, the expression of prolyl-hydroxylase domain (PHD)-2 and PHD-3 was found to be elevated in VVs compared with NVVs. Exposure of VV and NVV organ cultures to hypoxia or DMOG was associated with increases in HIF-1α and HIF-2α protein and HIF target gene expression compared with normoxia only. The study concluded, we believe for the first time, an increased activation of the HIF pathway, with upregulation of the expression of HIF-1α and HIF-2α transcription factors, and HIF target genes, in VVs compared with NVVs. Exposure of VVs and NVVs to hypoxic conditions was associated with increased expression of HIF-1α and HIF-2α protein and HIF target genes. The data suggest that the HIF pathway may be associated with several pathophysiologic changes in the VV wall, and that hypoxia may be a feature contributing to VV pathogenesis.

Bnip3 functions as a mitochondrial sensor of oxidative stress during myocardial ischemia and reperfusion

Bcl-2/adenovirus E1B 19-kDa protein-interacting protein 3 (Bnip3) is a member of the Bcl-2 homology domain 3-only subfamily of proapoptotic Bcl-2 proteins and is associated with cell death in the myocardium. In this study, we investigated the potential mechanism(s) by which Bnip3 activity is regulated. We found that Bnip3 forms a DTT-sensitive homodimer that increased after myocardial ischemia-reperfusion (I/R). The presence of the antioxidant N-acetylcysteine reduced I/R-induced homodimerization of Bnip3. Overexpression of Bnip3 in cells revealed that most of exogenous Bnip3 exists as a DTT-sensitive homodimer that correlated with increased cell death. In contrast, endogenous Bnip3 existed mainly as a monomer under normal conditions in the heart. Screening of the Bnip3 protein sequence revealed a single conserved cysteine residue at position 64. Mutation of this cysteine to alanine (Bnip3C64A) or deletion of the NH2-terminus (amino acids 1-64) resulted in reduced cell death activity of Bnip3. Moreover, mutation of a histidine residue in the COOH-terminal transmembrane domain to alanine (Bnip3H173A) almost completely inhibited the cell death activity of Bnip3. Bnip3C64A had a reduced ability to interact with Bnip3, whereas Bnip3H173A was completely unable to interact with Bnip3, suggesting that homodimerization is important for Bnip3 function. A consequence of I/R is the production of reactive oxygen species and oxidation of proteins, which promotes the formation of disulfide bonds between proteins. Thus, these experiments suggest that Bnip3 functions as a redox sensor where increased oxidative stress induces homodimerization and activation of Bnip3 via cooperation of the NH2-terminal cysteine residue and the COOH-terminal transmembrane domain.

BNIP-3: A novel candidate for an intrinsic depression-related factor found in NG108-15 cells treated with Hochu-ekki-to, a traditional oriental medicine, or typical antidepressants

Wakan-yaku is a type of Japanese and Sino traditional, systematized medical care that has been practiced for hundreds of years. To search for novel intrinsic factors related to the action of antidepressants, we used Hochu-ekki-to (HET), a Wakan-yaku medicine with antidepressive effects. First, we verified the quality of the HET by three-dimensional high-performance liquid chromatography and a cytotoxicity check in NG108-15 cells. We performed a DNA microarray analysis of the gene expression in cells treated with 50 μ/ml HET for more than 20 days. HET enhanced the expression of 125 (2.9%) genes and decreased the expression of 255 (6.0%) genes among the 4277 genes that were tested. The concentration-dependent increase in the expression of BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP-3) mRNA was particularly remarkable. A concentration-dependent increase in the expression of BNIP-3 mRNA was also observed when cells were treated with imipramine, mianserin, or milnacipran. These results suggest that BNIP-3 is a candidate for an intrinsic factor related to antidepressive effects and that Wakan-yaku theory may be useful for the identification of other intrinsic functional molecules.