Proteomics reveals lowering oxygen alters cytoskeletal and endoplasmatic stress proteins in human endothelial cells.
A proteomic approach was applied to explore the signalling pathways elicited by lowering O(2) in endothelial cells. Endothelial cells isolated from native umbilical cords were subjected to 21, 5, or 1% O(2) for 24 h. 2-D PAGE was performed and candidate proteins were identified using LC-MS/MS. Lowering of O(2) from 21 to 5% induced upregulation of cofilin-1, cyclophilin A, tubulin and tubulin fragments, a fragment of glucose-regulated protein 78 (Grp78) and calmodulin. The upregulation of Grp78 suggested that ER stress proteins were altered and indeed Grp94 and caspase 12 expression were increased in cells exposed to 5% O(2). The presence of ER stress is also supported by findings of blunted caffeine-evoked ER calcium release in cells exposed to 5 and 1% O(2). Exposure to 1% O(2) caused increases in cofilin-1, cyclophilin A, and caspase 12 as well as a decrease of beta-actin, but it did not alter the expression of calmodulin, tubulin, Grp78, and Grp94. Incubation with CoCl(2), a stabilizer of the hypoxia-inducible factor, increased the expression of several of the proteins. The present investigations reveal that lowering O(2), probably in part through hypoxia-inducible factor, alter the expression of a series of proteins mainly involved in cytoskeletal changes (e.g. cofilin-1, tubulin, and beta-actin) and in ER stress/apoptosis (e.g. Grp78/94, caspase 12, and cyclophilin A).
Ostergaard
Louise
L
Department of Pharmacology, University of Aarhus, 8000 Aarhus C, Denmark.
Simonsen
Ulf
U
Eskildsen-Helmond
Yvonne
Y
Vorum
Henrik
H
Uldbjerg
Niels
N
Honoré
Bent
B
Mulvany
Michael J
MJ
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D053454:Caspase 12 / Q000378:metabolism; D002478:Cells, Cultured; D021984:Cyclophilin A / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005260:Female; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008562:Membrane Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism*; D011247:Pregnancy; D040901:Proteomics / Q000379:methods; D015398:Signal Transduction / Q000502:physiology; D014470:Umbilical Cord / Q000166:cytology
D002147:Calmodulin; D003598:Cytoskeletal Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; C497442:HIF1A protein, human; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008562:Membrane Glycoproteins; C053387:endoplasmin; D053454:Caspase 12; D021984:Cyclophilin A; D010100:Oxygen; D002118:Calcium