Effect of TNF-α on human ARPE-19-secreted proteins

Purpose: 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-α (TNF-α) 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 ¹³C₆-Arg and ¹⁵N₂, ¹³C₆-Lys while the other set of cells was grown in unlabeled medium. The fully labeled cells were then treated with TNF-α, 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-α treated cells versus untreated cells. To validate the data, we performed a reverse experiment in which unlabeled cells were treated with TNF-α 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-α 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-α 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).