Nuclear localisation of cytosolic phospholipase A2-α in the EA.hy.926 human endothelial cell line is proliferation dependent and modulated by phosphorylation

Abstract

Cytosolic phospholipase A2-α (cPLA2-α) is a calcium-sensitive enzyme involved in receptor-mediated eicosanoid production. In resting cells, cPLA2-α is present in the cytosol and nucleus and translocates to membranes via its calcium-dependent lipid-binding (CaLB) domain following stimulation. cPLA2-α is also regulated by phosphorylation on several residues, which results in enhanced arachidonic acid release. Little is known about the factors controlling the nuclear localisation of cPLA2-α. Here the nuclear localisation of cPLA2-α in the EA.hy.926 human endothelial cell line was investigated. Nuclear localisation was dependent on proliferation, with subconfluent cells containing higher levels of nuclear cPLA2-α than contact-inhibited confluent or serum-starved cells. The broad-range protein kinase inhibitor staurosporine caused a decrease in the nuclear level of cPLA2-α, whereas the protein phosphatase inhibitor okadaic acid increased the level of nuclear cPLA2-α. Using inhibitors for specific mitogen-activated protein (MAP) kinases, both p42/44MAPK and p38MAPK were shown to be important in modulating nuclear localisation. Finally, inhibition of nuclear import and export using Agaricus bisporus lectin and leptomycin B, respectively, demonstrated that cPLA2-αcontains functional nuclear localisation and export signals. Thus we have identified a novel mode of regulation of cPLA2-α. This,together with the increasing body of evidence supporting the role of nuclear lipid second messengers in gene expression and proliferation, may have important implications for controlling the growth of endothelial cells in angiogenesis and tumour progression.

Release of Arachidonic Acid by Stimulation of Opsonic Receptors in Human Monocytes

“…This agrees with similar properties assigned to the structurally related Fc⑀RI expressed in mast cells, the cross-linking of which and the ensuing activation of the MAP kinase/cytosolic phospholipase A 2 route lead to AA release (28). However, this finding is at variance with our previous studies using the THP-1 cell line (19), points to the existence of important differences in the behavior of peripheral blood monocytes and monocytic cell lines, and suggests the existence of a variety of factors modulating the availability of AA and its likely influence by in vitro growing and cell division (29). Addressing the production of lipid mediators by Ab-driven mechanisms is a subject of pathophysiological relevance in view of the role played by these compounds in immune-mediated tissue injury, but the present findings may also be relevant to understand the process of maturation of monocyte-derived dendritic cells mediated by Fc␥R (30,31), in view of the role played by cPLA 2 in dendritic cell differentiation and function (32).…”

Cytoplasmic Phospholipase A2 Deletion Enhances Colon Tumorigenesis

“…On the one hand, the enzymatic actions of cPLA 2 provide arachidonic acid for the synthesis of eicosanoids, many of which can promote tumor cell growth, proliferation, and angiogenesis. However, there is considerable evidence that intracellular pools of arachidonic acid released by cPLA 2 play a key role in ceramide-mediated signal transduction that culminates in apoptosis (24,25,28,35). During tumorigenesis, arachidonic acid homeostasis is often altered, and the tightly controlled balance between apoptotic and proliferative signals is potentially disrupted.…”

“…cPLA 2 is the most well characterized isoform and the only one that is widely expressed in the colon (20). cPLA 2 is activated both by Ca 2+ binding and mitogen-activated protein kinase phosphorylation (23,24). In resting cells, cPLA 2 is present in the cytosol and upon stimulation by Ca 2+ binding, translocates to membranes where it is positioned adjacent to the COXs within the nuclear envelope and endoplasmic reticular membrane, thus serving as a major source of arachidonic acid substrate for PG production (24).…”

“…We recently reported that tumor necrosis factor (TNF)-a-induced apoptosis was attenuated in cultured mouse colonocytes in which cPLA 2 expression was reduced by treatment with antisense oligos (26). Several other studies have shown that when TNF-a induces cPLA 2 , arachidonic acid is released, triggering activation of sphingomyelinases to generate ceramide, which in turn signals G 0 -G 1 cell cycle arrest and apoptosis (24,(27)(28)(29)(30). The direct dose-dependent stimulation of sphingomyelinase activity by arachidonic acid has been previously established in a cell-free system (28).…”

The CRM1 Nuclear Export Receptor Controls Pathological Cardiac Gene Expression

“…Components of these antihypertrophic networks include the genes encoding glycogen-synthase kinase 3␤ (1, 23), A20 (11), phospholipase A2 (24), cyclic-GMP-dependent protein kinase (15,26,62), and c-Jun N-terminal kinase (36,53). Notably, GSK-3␤, phospholipase A2 and cyclic-GMP-dependent protein kinase have all been shown to shuttle between the nucleus and the cytoplasm (18,20,23). Thus, it is conceivable that LMBmediated nuclear retention of these signaling molecules con- FIG.…”