Troglitazone and 15-deoxy-delta(12,14)-prostaglandin J2 inhibit shear-induced coupling factor 6 release in endothelial cells.
OBJECTIVE
We previously showed that mitochondrial coupling factor 6 (CF6), an endogenous inhibitor of prostacyclin synthesis and a vasoconstrictor, is present on the surface of human umbilical vein endothelial cells (HUVEC) and is released outside of the cells by shear stress. We investigated the intracellular signaling mechanism for shear-induced release of CF6 in HUVEC and the effects of troglitazone and 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), both peroxisome proliferator-activated receptor (PPAR)-gamma ligands, on it.


RESULTS
The release and gene expression of CF6 in HUVEC were enhanced by shear stress at 25 dyn/cm2, measured by radioimmunoassay and real-time RT-PCR, respectively. The intracellular content of CF6 was decreased after exposure to shear stress at 25 dyn/cm2. Transfection experiments with deletional and mutational CF6 promoter constructs, and with dominant negative mutant IkappaB kinase alpha (K44M) demonstrated that shear-induced CF6 transcription was dependent on nuclear factor-kappa B (NF-kappaB) activation. Pretreatment with troglitazone or 15d-PGJ2 inhibited the shear-induced release and gene expression of CF6, whereas fenofibric acid, a PPAR-alpha ligand, had no influence on them. Western blot and immunostaining showed that troglitazone and 15d-PGJ2 inhibited the shear-induced, reactive oxygen species (ROS)-mediated activation of NF-kappaB at the level of IkappaB protein.


CONCLUSIONS
The shear-induced gene expression and release of CF6 in HUVEC are mediated by the ROS-related activation of NF-kappaB signaling pathway. Troglitazone and 15d-PGJ2 inhibit them at the IkappaB protein level.
Tomita
Hirofumi
H
The Second Department of Internal Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562 Japan.
Osanai
Tomohiro
T
Toki
Tsutomu
T
Sasaki
Satoko
S
Maeda
Naotaka
N
Murakami
Reiichi
R
Magota
Koji
K
Yasujima
Minoru
M
Okumura
Ken
K
D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D002839:Chromans / Q000494:pharmacology*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D008024:Ligands; D008856:Microscopy, Fluorescence; D025261:Mitochondrial Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D010086:Oxidative Phosphorylation Coupling Factors / Q000235:genetics / Q000378:metabolism*; D047495:PPAR gamma / Q000378:metabolism*; D015230:Prostaglandin D2 / Q000031:analogs & derivatives* / Q000494:pharmacology; D011863:Radioimmunoassay; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013314:Stress, Mechanical; D045162:Thiazolidinediones / Q000494:pharmacology*; D000077288:Troglitazone
C477819:15-deoxyprostaglandin J2; D002839:Chromans; D020672:I-kappa B Proteins; D008024:Ligands; D016328:NF-kappa B; D010086:Oxidative Phosphorylation Coupling Factors; D047495:PPAR gamma; D017382:Reactive Oxygen Species; D045162:Thiazolidinediones; C000628217:F(6) ATPase; D025261:Mitochondrial Proton-Translocating ATPases; D000077288:Troglitazone; D015230:Prostaglandin D2