Differential Roles for Cytosolic and Microsomal Ca²⁺-Independent Phospholipase A₂in Cell Growth and Maintenance of Phospholipids

Abstract

Physiological roles of microsomal (iPLA 2 ␥) and cytosolic (iPLA 2 ␤) Ca 2ϩ -independent phospholipase A 2 were determined in two different epithelial cell models. R-and S-enantiomers of the iPLA 2 inhibitor bromoenol lactone (BEL) were isolated and shown to selectively inhibit iPLA 2␥ and iPLA 2␤ , respectively. The effect of these enantiomers on cell growth was assessed in human embryonic kidney 293 and Caki-1 cells using 3-(4-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT). S-BEL (0 -5.0 M) decreased MTT staining 35% after 24 h compared with control cells, whereas treatment with either R-BEL or R/S-BEL induced 15% decreases. Neither R-BEL nor S-BEL induced cell death as determined by annexin V and propidium iodide staining. Transfection of cells with iPLA 2 ␤ siRNA reduced MTT staining approximately 35%, whereas transfection of cells with iPLA 2 ␥ siRNA only decreased MTT staining 10 to 15% compared with control cells. The effect of iPLA 2 ␤ and iPLA 2 ␥ siRNA on cell number and protein was also determined, and iPLA 2 ␤ siRNA decreased cell number and protein 25% compared with control cells. In contrast, iPLA 2 ␥ siRNA decreased cell number, but not cellular protein, compared with control cells. Selective inhibition of iPLA 2 ␤, but not iPLA 2 ␥, decreased several arachidonic acid-containing phospholipids, including 16:1-20:4, 16:0-20:4, 18:1-20:4, and 18:0-20:4 phosphatidylcholine, showing that the ability of iPLA 2 ␤ inhibitors to decrease cell growth correlates with their ability to decrease arachidonic acid-containing phospholipids. These data show that iPLA 2 ␤ inhibition results in greater decreases in cell growth and proliferation than iPLA 2 ␥, identifies specific phospholipids whose expressions are differentially regulated by iPLA 2 ␤ and iPLA 2 ␥, and suggests novel roles for iPLA 2 ␤ in cell growth.

Inhibition of Ca2+-Independent Phospholipase A2 Decreases Prostate Cancer Cell Growth by p53-Dependent and Independent Mechanisms

“…Inhibition of iPLA 2 decreases proliferation in several types of cancer and noncancerous cells (Saavedra et al, 2006;Zhang et al, 2006). Decreased INS-1 cell growth correlated to p53-mediated G 1 arrest .…”

“…We previously demonstrated that BEL inhibits iPLA 2 activity with an IC 50 of 1 to 2.5 M (Kinsey et al, 2005;Saavedra et al, 2006). Furthermore, we and others (Jenkins et jpet.aspetjournals.org onstrated the selectivity of R-BEL for iPLA 2 ␥ and S-BEL for iPLA 2 ␤.…”

“…However, it recently has drawn attention as a cell growth regulator because of its possible role in insulin secretion (Ramanadham et al, 1999), eicosanoid metabolism (Akiba et al, 1998), phospholipid remodeling , apoptosis (Atsumi et al, 2000;Cummings et al, 2004), vascular relaxation (Seegers et al, 2002), and adipogenesis (Su et al, 2004). Recent studies also suggest that iPLA 2 mediates cell proliferation in HEK293 (Saavedra et al, 2006), human insulinoma, colon cancer (Bao et al, 2006;Zhang et al, 2006), and ovarian cancer cells (Song et al, 2007). It is not known whether iPLA 2 mediates prostate cancer cell growth.…”

Calcium-independent phospholipases A2 and their roles in biological processes and diseases

“…The ability of iPLA 2 ␤ to promote cell proliferation becomes prominent in the context of tumorigenesis. Several in vitro studies reveal higher expression of iPLA 2 ␤ in stimulated immortal cell lines and that chemical inhibition or siRNAs targeted against iPLA 2 ␤ reduces proliferation and promotes apoptosis of the cells ( 97, 108,125,[356][357][358][359][360][361][362][363][364]. Subsequent studies targeting specifi c cancers suggest that iPLA 2 ␤ promotes cancer cell growth via signal transduction pathways involving epidermal growth factor receptors, MAPKs, E3 ubiquitin-protein ligase mdm2, tumor suppressor protein p53, and cell cycle regulator p21 (365)(366)(367).…”

Complement-mediated Activation of Calcium-independent Phospholipase A2γ

“…Therefore, iPLA 2 plays a housekeeping role by facilitating phospholipid remodeling (19 -21). Inhibition of iPLA 2 in HEK293 and INS-1 cells altered the amounts of several phospholipids and resulted in decreased cell growth and p53 activation (22)(23)(24). During oxidative stress, an ER-associated iPLA 2 in renal proximal tubular cells (iPLA 2 ␥) recognizes, cleaves, and removes oxidized phospholipids from the ER membrane (25); thus, iPLA 2 ␥ may act to repair or prevent lipid peroxidation during oxidative stress (25).…”