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S.G. Barreiro, M. Kolko, N.R. Christoffersen, N.G. Bazan; Secretory Phospholipases A2 in Human RPE Cells: Novel Intercellular Messengers . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2284.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Phospholipases A2 (PLA2) release polyunsaturated fatty acids in the RPE cells. To date most studies have been concerned with the cytosolic PLA2 (cPLA2). In addition there are secretory PLA2s (sPLA2) that bind to receptors. There are several different groups of sPLA2s that have been characterized. Here we report the cloning and characterization of novel sPLA2-IB and X in human RPE cells. Methods: Based on the sequence of human PLA2 group IB and X in pancreas [Gispert et al., (1993) Nat Genet 4: 295-299] and spleen [Cuppillard et al., (1997) J Biol Chem 272: 15745-15752] we designed primers to identify the sPLA2s in human ARPE-19 cells. Cells were grown in 6-well plates, RNA was isolated, and cDNA was produced and used for PCR cloning to identify the two novel subtypes of sPLA2. ARPE cells were serum-starved 6 hr prior to addition of TNF-α. Cells were incubated overnight and TNF-α was removed by addition of new medium. Cells were kept at 37°C until harvesting. Results: Using PCR cloning we identified sPLA2-IB and X in human ARPE-19 cells. We confirmed the cloned PCR fragment by sequencing and designed primers for real-time PCR based on our cloned sequence. By real-time PCR we further showed a 5.5-fold induction of sPLA2-IB when RPE cells were exposed to TNF-α. Conclusions: This study is the first to identify specific novel sPLA2s in human RPE cells and suggests a possible significance of these enzymes in retinal repair and remodeling. TNF-α expression in the RPE cells is involved in macular degeneration, and we speculate that novel sPLA2s may be novel intercellular messengers that may be significant in the pathophysiology of diseases such as macular degeneration.
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