Translocation of the 85-kDa Phospholipase A2 from Cytosol to the Nuclear Envelope in Rat Basophilic Leukemia Cells Stimulated with Calcium Ionophore or IgE/Antigen

Abstract

The rat mast cell line RBL-2H3.1 contains an 85-kDa cytosolic phospholipase A2 (cPLA2) that is very likely involved in liberating arachidonate from membrane phospholipid for the synthesis of eicosanoids following stimulation with either calcium ionophore or IgE/antigen. In this study, the intracellular location of cPLA2 was determined using immunofluorescence microscopy and immuno-gold electron microscopy. In nonstimulated cells, cPLA2 is distributed throughout the cytosol and is excluded from the nucleoplasm. Following cell activation with calcium ionophore, most of the cPLA2 translocates to the nuclear envelope, and the enzyme remains there during the entire period that ionophore is present. With IgE/antigen stimulation for 5 min, approximately 20-30% of the cPLA2 translocates to the nuclear envelope, and after 30 min of stimulation, most of the enzyme returns to the cytosol. Measurement of intracellular calcium using the dye Fura-2/AM shows that the level of calcium rises immediately after antigen is added, remains high for about 30 s, and then declines back to resting levels. Activation with calcium ionophore produces a 10-fold larger release of arachidonate than does stimulation with IgE/antigen. Thus, the results suggest that the extent of membrane binding of cPLA2 correlates with the release of arachidonate and that the site of arachidonate liberation is the nuclear envelope where many of the enzymes that oxygenate this fatty acid are located.

Cytoplasmic phospholipase A2 alpha overexpression in stromal cells is correlated with angiogenesis in human colorectal cancer

“…38 Although, an increased protein expression does not necessarily indicate an important activation or increased function, the moderate perinuclear cPLA2 distribution seen by confocal microscopy could be related to cytoplasmic PLA2 activation. 39 Since our approach is based on immunohistochemistry and image analysis in human lesions, we could not directly demonstrate a high cytoplasmic PLA2 activity or high prostaglandin production. We thus wanted to determine if cytoplasmic PLA2 protein overexpression in stromal cells could be linked with a biological effect known to be regulated in colorectal cancer by COX-2-induced prostaglandin production.…”

A rise in nuclear calcium translocates annexins IV and V to the nuclear envelope

“…Using microscopy methods, Glover and co-workers [5] showed in the rat mast cell line RBL-2H3 that cPLA2 translocated to the nuclear envelope following A23187 treatment. They suggested that this translocation might be due to a putative receptor or to a specific phospholipid composition of the nuclear membrane.…”

“…Although the translocation of nuclear Anxs to the inner side of the nuclear envelope during a rise in nucleoplasmic calcium is not unexpected, our results show that even cytosolic annexins are relocalized to the nuclear membrane. Similarly, cPLA2, that is excluded from the nucleoplasm in resting cells, translocates to the nuclear envelope during A23187 treatment [5]. The permeability of the nuclear envelope towards Ca 2+ has been extensively debated for the last few years, but recent data led to the conclusion that release from Ca 2+ stores in or around the nuclear envelope is directed into the nucleoplasm from where it can diffuse out through the permeable nuclear pore complex and thus increase the cytosolic [Ca 2+] in the immediate vicinity of the nuclear envelope [11].…”

“…Finally, to our knowledge, the translocation described herein and by Barwise and Walker [10] constitutes the first evidence of a Ca 2+-regulated interaction of Anxs with cell membranes in situ, although it occurs following stimulation with a Ca 2+ ionophore. Interestingly, relocalization of cPLA2 to the nuclear envelope was initially found during A23187 treatment [4], but a partial translocation has been recently reported during IgE/antigen stimulation of mast cells [5]. It will be most interesting to determine whether Anxs are also translocated under similar stimulation.…”

“…E-mail: raynal@cict.fr escence microscopy to study the intracellular localization of various annexins in human fibroblasts stimulated by various Ca 2+ agonists. We have observed that Anxs IV and V selectively translocate to the nuclear envelope during stimulation with calcium ionophore A23187, similarly to the 85-kDa cytosolic phospholipase A2 (cPLA2) [4,5]. To gain insight into the mechanism of this translocation, we have studied the intracellular calcium concentration ([CaZ+]) by confocal fluorescence microscopy of cells loaded with Ca2+-sensitive probes.…”