Maintenance of Bad phosphorylation prevents apoptosis of rat hepatic sinusoidal endothelial cells in vitro and in vivo.
To elucidate the mechanism of apoptosis of liver sinusoidal endothelial cells (SECs), we examined the phosphorylation status of Bad and its upstream signaling molecules during apoptosis in culture and after ischemia-reperfusion injury. Rat SECs were isolated by the immunomagnetic method, and 2 days after culture, most SECs underwent apoptosis, which was associated with decreased tyrosine phosphorylation of cellular proteins. Addition of orthovanadate (OV), a protein tyrosine phosphatase inhibitor, sustained cellular protein phosphorylation and strongly inhibited apoptosis. Bad was dephosphorylated at Ser-112 and Ser-136 during apoptosis, but the phosphorylation status of Bad was maintained in the presence of OV. OV activated the Akt, extracellular signal-regulated protein kinase, and p38 mitogen-activated protein kinase pathways, which are involved in Bad phosphorylation. In the absence of OV, depletion of Bad by RNA interference conferred resistance to apoptosis. Hepatic injury after ischemia-reperfusion was alleviated by OV treatment, with significant inhibition of SEC apoptosis. SEC apoptosis in vivo was associated with dephosphorylation of Bad, Akt, and extracellular signal-regulated protein kinase, which was blocked by OV treatment. Our data suggest that maintenance of Bad phosphorylation is important in the prevention of SEC apoptosis and that the anti-apoptotic property of OV might have therapeutic utility.
Department of Pathology and Immunology, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan.
Naoto
N
Ohi
Yuji
Y
Nishikawa
Takuo
T
Tokairin
Yohei
Y
Yamamoto
Yuko
Y
Doi
Yasufumi
Y
Omori
Katsuhiko
K
Enomoto
D048948:14-3-3 Proteins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D008099:Liver / Q000098:blood supply / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051058:Oncogene Protein v-akt / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D011916:Rats, Inbred F344; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000473:pathology; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology; D051025:bcl-Associated Death Protein / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D048948:14-3-3 Proteins; D004791:Enzyme Inhibitors; D051025:bcl-Associated Death Protein; D014638:Vanadates; D014443:Tyrosine; D012694:Serine; D051058:Oncogene Protein v-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C505410:Casp3 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases