Regulation of Cytosolic Phospholipase A2 (cPLA2α) and Its Association with Cell Proliferation in Human Lens Epithelial Cells

Cytosolic phospholipase A2α (cPLA2α) belongs to the large phospholipase A2 (PLA2) family that is widely involved in various fundamental cellular physiological functions, including neurochemical processes, normal brain function, regulation of protein kinase C and modulation of ion channels.1,2 cPLA2α is widely distributed in mammalian tissues and can be activated by extracellular stimuli such as growth factors, cytokines, neurotransmitters, or endotoxins,2 and selectively hydrolyzes glycerophospholipids containing arachidonic acid (AA) at the sn-2 position. The released AA can be further metabolized to leukotrienes, prostaglandins, or platelet-activating factor for various biological needs.3–5 The subcellular localization and target of cPLA2α may vary depending on the cell type; however, cPLA2α requires cellular calcium binding at the C2 domain to be translocated to the membrane for its catalytic action. For instance, in endothelial cells, cPLA2α is mobilized from the cytosol to the perinuclear region (endoplasmic reticulum, Golgi, and nuclear envelope) where the AA produced is metabolized to eicosanoids6 while in neutrophils and granulocytes, cPLA2α is recruited to plasma membrane in which the AAs released are needed for the generation of reactive oxygen species (ROS) from NADPH oxidase. Translocating cPLA2α to the membrane is an important step as it allows cPLA2α to be phosphorylated and activated by ERK so that it can interact with its phospholipid substrate on the membrane to release AA.7 Serine residues in positions 228, 437, 454, 505, and 727 of cPLA2α have been identified to be the sites of phosphorylation. In particular, the site at Ser 228 is known to control cPLA2α activity. Calcium ion (Ca2+) is an important biological regulator, which is broadly involved in various cellular signaling pathways of physiologic or pathologic events.8 The normally low intracellular level of calcium can be enriched by mobilization of the internal store in the endoplasmic reticulum (ER) or from the extracellular medium. Typically, stimulation of G-protein coupled receptor or tyrosine kinase receptors by outside stimuli can activate the isoforms of phospholipase C (PLC), which in turn hydrolyzes phosphatidyl inositol 4,5-tri-phosphate (PIP2) to release the secondary messenger of inositol triphosphate (IP3). The subsequent binding of IP3 to the receptor on the ER membrane allows the calcium stored inside to be released into the cytoplasm. On the other hand Ca adenosine triphosphatase (ATPase) located on the ER membrane can pump calcium back into ER to replenish the store. Homeostasis of intracellular calcium is essential to the health of cells and tissues. For instance, it is known that calcium is needed in cell proliferation,9 however, too great an influx of calcium or disruption of the ER store can both lead to cell death and diseases.10–12 Recently, attention has been drawn to the relationship between Ca2+ and ROS with the demonstration that Ca2+ is required in many ROS-related cellular activities,13,14 one of which is cell proliferation. Sundaresan et al.15 first reported that platelet-derived growth factor (PDGF) mitogenic action is mediated by ROS generation. Bae et al.16 later showed that epidermal growth factor (EGF) signaling for cell proliferation was also regulated by ROS production. Therefore, redox signaling has been suggested in many cell types to be the key regulator for growth factor- and/or cytokine-induced cellular functions, including cell proliferation, migration, and wound healing. Recently ROS signaling has also been recognized as the essential regulator for PDGF function in the lens17 and EGF function in the cornea.18 In both lens and corneal epithelial cells, cPLA2α was shown to play a major role, as cPLA2α inhibition led to ceased ROS generation from NADPH oxidase,19 and subsequently halted cell proliferation, migration, and even wound healing in cells stimulated with PDGF or EGF.18 Even though cPLA2α is critical for growth factor-induced ROS generation in the lens epithelial cells,19 the detailed mechanism underlying cPLA2α activation and function remains unclear in the ocular tissues. While the cellular level of calcium, especially that in the calcium store in ER, appears to be closely associated with EGF-stimulated growth and survival of lens epithelial cells,20 little is known about any possible role played by calcium in the function of cPLA2α in ocular tissues. In the present study, we have investigated the significance of calcium ion in PDGF-induced ROS signaling by manipulating intracellular calcium level with ionomycin to increase external calcium influx, thapsigargin to release ER calcium stores, or a chelating agent to deplete calcium. We have examined the regulation of cPLA2α by using a stable dominant-negative cPLA2α (S228A) HLE B3 cell line, and a transient cell line with truncated C2 calcium binding domain (C2D) to examine their effects on PDGF signaling. Our results indicate that an intact and active cPLA2α as well as the presence of adequate intracellular calcium are essential for PDGF-elicited ROS production and cell proliferation. For simplicity, cPLA2α IV will be referred to as cPLA2 from this point on in this report.