S179D prolactin primarily uses the extrinsic pathway and mitogen-activated protein kinase signaling to induce apoptosis in human endothelial cells.
We have demonstrated that S179D prolactin (PRL) is potently antiangiogenic in vivo. Here, we examined apoptosis in human endothelial cells, using procaspase-8 and cytochrome c release as markers of the extrinsic and intrinsic pathways, respectively. Both pathways converge at caspase-3, which is responsible for cleavage of DNA fragmentation factor (DFF45). A 3-d incubation in 50 ng/ml S179D PRL quadrupled the number of early apoptotic cells; this effect was doubled at 100 ng/ml and became maximal at 500 ng/ml. DFF45 and procaspase 8 cleavage were detectable at 100 ng/ml. Cytochrome c, however, was unaffected until 500 ng/ml. The p21 increased at 24 h, whereas a change in p53 required both triple the time and higher doses. The p21 promoter activity was maximal at 50 ng/ml, whereas 500 ng/ml were required to see a significant change in the Bax promoter (a measure of p53 activity). Because S179D PRL and basic fibroblast growth factor (bFGF) have both been shown to activate ERK, the effect of S179D PRL on bFGF-induced ERK signaling was examined. S179D PRL blocked ERK phosphorylation in response to bFGF, whereas continued coincubation caused a delayed and prolonged activation of ERK. PD98059 inhibited this delayed activation of ERK and effects of S179D PRL on all measures except p53 levels or activity of the Bax promoter. We conclude that S179D PRL blocks bFGF-induced ERK signaling and yet uses ERK in a different time frame to elevate p21 and activate the extrinsic pathway. Prolonged incubations and high concentrations additionally activate the intrinsic pathway using an alternate intracellular signal.
Ueda
Eric K
EK
Division of Biomedical Sciences, University of California, Riverside, 92521, USA.
Lo
Hsin-Lung
HL
Bartolini
Paolo
P
Walker
Ameae M
AM
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D004396:Coloring Agents; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation; D042783:Endothelial Cells / Q000502:physiology*; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D008156:Luciferases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D010718:Phosphatidylserines / Q000378:metabolism; D011388:Prolactin / Q000502:physiology*; D011419:Propidium; D015398:Signal Transduction / Q000502:physiology*; D046509:Tissue Culture Techniques; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D017304:Annexin A5; D004396:Coloring Agents; D010718:Phosphatidylserines; D011419:Propidium; D011388:Prolactin; D045304:Cytochromes c; D004247:DNA; D008156:Luciferases; D020928:Mitogen-Activated Protein Kinases; D001616:beta-Galactosidase