Regulation of choline deficiency apoptosis by epidermal growth factor in CWSV-1 rat hepatocytes.
Previous studies show that acute choline deficiency (CD) triggers apoptosis in cultured rat hepatocytes (CWSV-1 cells). We demonstrate that prolonged EGF stimulation (10 ng/mL x 48 hrs) restores cell proliferation, as assessed by BrdU labeling, and protects cells from CD-induced apoptosis, as assessed by TUNEL labeling and cleavage of poly(ADP-ribose) polymerase. However, EGF rescue was not accompanied by restoration of depleted intracellular concentrations of choline, glycerphosphocholine, phosphocholine, or phosphatidylcholine. In contrast, we show that EGF stimulation blocks apoptosis by restoring mitochondrial membrane potential (Delta Psi(m)), as determined using the potential-sensitive dye chloromethyl-X-rosamine, and by preventing the release and nuclear localization of cytochrome c. We investigated whether EGF rescue involves EGF receptor phosphorylation and activation of the down-stream cell survival factor Akt. Compared to cells in control medium (CT, 70 micromol choline x 48 hrs), cells in CD medium (5 micromol choline) were less sensitive to EGF-induced (0-300 ng/mL x 5 min) receptor tyrosine phosphorylation. Compared to cells in CT medium, cells in CD medium treated with EGF (10 ng/mL x 5 min) exhibited higher levels of phosphatidylinositol 3-kinase (PI3K)-dependent phosphorylation of AktSer473. Inactivation of PI3K was sufficient to block EGF-stimulated activation of Akt, restoration of mitochondrial Delta Psi(m), and prevention of cytochrome c release. These studies indicate that stimulation with EGF activates a cell survival response against CD-apoptosis by restoring mitochondrial membrane potential and preventing cytochrome c release and nuclear translocation which are mediated by activation of Akt in hepatocytes.
Albright
Craig D
CD
Department of Nutrition, School of Public Health and School of Medicine, University of North Carolina, Chapel Hill, NC 27599-7461, USA.
da Costa
Kerry-Ann
KA
Craciunescu
Corneliu N
CN
Klem
Erich
E
Mar
Mei-Heng
MH
Zeisel
Steven H
SH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002794:Choline / Q000378:metabolism; D002796:Choline Deficiency / Q000378:metabolism / Q000473:pathology*; D045304:Cytochromes c / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D011916:Rats, Inbred F344; D014157:Transcription Factors / Q000378:metabolism
D014157:Transcription Factors; D004815:Epidermal Growth Factor; D045304:Cytochromes c; D066246:ErbB Receptors; D002794:Choline