Premium
Novel and Differential Roles of Ca 2+ ‐Independent Phospholipase A 2 Isoforms in Renal Cell Growth and Phospholipid Metabolism
Author(s) -
Cummings Brian,
Peterson Brianna,
Saavedra Geraldine
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1138-d
Subject(s) - mtt assay , phospholipid , staining , cell growth , chemistry , microbiology and biotechnology , transfection , phospholipase a2 , cytosol , cell culture , phosphatidylcholine , cell , phospholipase , biochemistry , biology , enzyme , membrane , gene , genetics
Recent studies demonstrate that human renal cells express both cytosolic (iPLA 2 β) and microsomal (iPLA 2 γ) Ca 2+ ‐independent phospholipase A 2 (iPLA 2 ). However, the roles of these iPLA 2 in renal cell physiology are not fully understood. The effect of R‐bromonenol lactone (R‐BEL, iPLA 2 γ inhibitor) and S‐BEL (iPLA 2 β inhibitor) on cell growth was assessed in HEK293 and Caki‐1 (human renal epithelial) cells using 3‐(4, dimethylthiazolyl‐2)‐2, 5‐diphenyltetrazolium bromide (MTT) staining. Treatment of cells with S‐BEL (0‐5.0 μM) decreased MTT staining 35% after 48 hr. In contrast, R‐BEL (0–5.0 μM) only decreased MTT staining 15%, compared to control cells. Transfection of cells with iPLA 2 β siRNA reduced MTT staining 35%, while iPLA 2 γ siRNA only decreased MTT staining 10–15%. siRNA against iPLA 2 β also resulted in 25% decreases in cell number and protein, while siRNA against iPLA 2 γ only slightly decreased cell number, and did not alter cellular protein. To determine the roles of iPLA 2 β and γ in phospholipid metabolism the effect of R‐ and S‐BEL on phospholipid profiles was determined using electrospray ionization‐mass spectrometry. Treatment of cells with R‐BEL alone decreased the expression of 14:0–16:0 phosphatidylcholine (PtdCho), 16:0–20:4 PtdCho and increased the expression of 16:0–20:1 PtdCho. In comparison, treatment of cells with S‐BEL increased the expression of 16:1–20:4 PtdCho and 20:1–22:4 plasmenylcholine. These data demonstrate a novel role for iPLA 2 β and γ in renal cell growth and suggest that these isoforms have differential roles in phospholipid metabolism. Supported by a Georgia Cancer Coalition Distinguished Scholar Award.