Effect of TNF-alpha on human ARPE-19-secreted proteins.
OBJECTIVE
To identify cytokine-induced changes in the secretome of human retinal pigment epithelial (RPE) cells and their potential implication in age-related macular degeneration pathogenesis.


METHODS
Stable isotope labeling by amino in cell culture (SILAC) was used in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) to measure differential protein secretion from tumor necrosis factor-alpha (TNF-alpha) treated ARPE-19 versus untreated ARPE-19 cells. Typically, one set of cells was subcultured in a medium in which Arg and Lys were replaced by (13)C(6)-Arg and (15)N(2,) (13)C(6)-Lys while the other set of cells was grown in unlabeled medium. The fully labeled cells were then treated with TNF-alpha, while unlabeled cells were left untreated. Spent media from both treated and untreated cells were collected, mixed at 1:1 ratio, and processed for LC-MS/MS analysis. Labeled and unlabeled peptide pairs were identified and their intensities were used to determine protein ratios in TNF-alpha treated cells versus untreated cells. To validate the data, we performed a reverse experiment in which unlabeled cells were treated with TNF-alpha while labeled cells were kept untreated.


RESULTS
A total of 146 proteins were identified as putatively secreted proteins in the spent medium of ARPE-19 cells and only six among these were differentially secreted following TNF-alpha treatment. Secretion of complement 3 and sulfhydryl oxidase-1 was increased by twofold, fibronectin by 1.7 fold, plasminogen activator inhibitor 1 by 1.9 fold and syndecan-4 by 4.35 fold while secretion of trans-golgi network protein-2 was decreased by twofold.


CONCLUSIONS
TNF-alpha modulates secretion of specific proteins in ARPE-19 cells. These proteins are involved in pathways relevant to AMD pathogenesis (e.g., extracellular matrix remodeling, complement pathway, and angiogenesis).
An
Eunkyung
E
Center for Genetic Medicine, Children's National Medical Center, Washington, DC 20010, USA.
Gordish-Dressman
Heather
H
Hathout
Yetrib
Y
D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D005136:Eye Proteins / Q000378:metabolism*; D006801:Humans; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D005136:Eye Proteins; D014409:Tumor Necrosis Factor-alpha