Nitrogen dioxide-induced expression of a 78 kDa protein in pulmonary artery endothelial cells.
Exposure to nitrogen dioxide (NO2) activates signal transduction in cultured pulmonary artery endothelial cells (PAEC). We examined whether NO2-induced activation of signal transduction results in increased expression of proteins in PAEC. Exposure to 5 ppm NO2 for 4, 12, and 24 h had no significant effect on total protein synthesis. However, two-dimensional gel electrophoresis of [35S]-methionine-labeled PAEC exposed to NO2 for 24 h, but not 4 and 12 h, demonstrated increased synthesis of several proteins including a two- to five-fold increase of some proteins with molecular masses of 47, 64, 78, and 105 kDa compared to controls. N-terminal amino acid sequencing and immunodetection analysis identified the 78 kDa protein as 78 kDa glucose-regulated protein (GRP-78). Induction of GRP-78 by NO2 exposure was regulated at the transcriptional level, and the induction required de novo protein synthesis. Exposure to NO2 for 24 h also significantly (p < .05) decreased glycosylation of proteins in PAEC. Exposure of cell monolayers to tunicamycin, an inhibitor of protein glycosylation, mimicked the effect of NO2 exposure on expression of GRP-78. Increased expression of GRP-78 was also detected when cell monolayers were exposed to the calcium ionophore A 23187, to 2-deoxyglucose, or to glucose-free medium, which are also known to cause perturbations in protein glycosylation. These results demonstrate that exposure to NO2 increases expression of a number of proteins including GRP-78 in PAEC. Increased expression of GRP-78 in NO2-exposed cells appears to be associated with inhibition of glycosylation or through coordinated alterations in metabolic events that lead to inhibition of protein glycosylation.
Li
Y D
YD
Division of Pulmonary Medicine, University of Florida College of Medicine, Gainesville, USA.
Patel
J M
JM
Block
E R
ER
D000595:Amino Acid Sequence; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D003847:Deoxyglucose / Q000494:pharmacology; D000091342:Endoplasmic Reticulum Chaperone BiP; D004730:Endothelium, Vascular / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects*; D006031:Glycosylation; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones / Q000096:biosynthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009585:Nitrogen Dioxide / Q000494:pharmacology*; D011651:Pulmonary Artery; D015398:Signal Transduction / Q000187:drug effects; D013552:Swine; D014415:Tunicamycin / Q000494:pharmacology
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D014415:Tunicamycin; D003609:Dactinomycin; D000001:Calcimycin; D003513:Cycloheximide; D003847:Deoxyglucose; D009585:Nitrogen Dioxide