Tumor necrosis factor alpha as an endogenous stimulator for circulating coupling factor 6.
OBJECTIVE
We recently showed that mitochondrial coupling factor 6 (CF6) is present as a pressor substance and a prostacyclin inhibitor in systemic circulation. However, the regulation mechanism for circulating CF6 is unknown. We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in the generation and release of CF6.


RESULTS
We used two kinds of cells, human umbilical vein endothelial cells (HUVEC) and ECV-304. The concentration of CF6 in the medium increased with time in both ECV-304 and HUVEC. Treatment of ECV-304 and HUVEC with TNF-alpha enhanced the release of CF6 in a dose-dependent manner concomitantly with the decrease in CF6 content in the mitochondria at 24 h. The released CF6 was characterized to be an active full-length peptide by Western blot. The ratio of CF6 to GAPDH mRNA, measured by real-time polymerase chain reaction, was 1.7 fold increased at 1 h after exposure to TNF-alpha in ECV-304 and HUVEC. This enhanced gene expression and release was blocked or suppressed by 70% by stable transfection of dominant negative mutant I kappa B kinase alpha whose efficacy was confirmed by blockade of translocation of NF-kappa B p65 protein and of degradation of I kappa B alpha protein. Flow cytometry analysis revealed that the cell surface-associated CF6 was significantly increased at 24 h after TNF-alpha in a dose-dependent manner.


CONCLUSIONS
TNF-alpha stimulates the gene expression of CF6 via activation of NF-kappa B signaling pathway, and promotes the release of CF6 from ECV-304 and HUVEC.
Sasaki
Satoko
S
The Second Department of Internal Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.
Osanai
Tomohiro
T
Tomita
Hirofumi
H
Matsunaga
Toshiro
T
Magota
Koji
K
Okumura
Ken
K
D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D002478:Cells, Cultured; D015237:Dinoprost / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D020672:I-kappa B Proteins / Q000032:analysis / Q000378:metabolism; D008856:Microscopy, Fluorescence; D025261:Mitochondrial Proton-Translocating ATPases / Q000097:blood / Q000235:genetics*; D016328:NF-kappa B / Q000378:metabolism*; D010086:Oxidative Phosphorylation Coupling Factors / Q000097:blood / Q000235:genetics*; D011863:Radioimmunoassay / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
C078129:6-keto-prostaglandin F2alpha; D020672:I-kappa B Proteins; D016328:NF-kappa B; D010086:Oxidative Phosphorylation Coupling Factors; D014409:Tumor Necrosis Factor-alpha; D015237:Dinoprost; C000628217:F(6) ATPase; D025261:Mitochondrial Proton-Translocating ATPases