Cytosolic phospholipase A2ε drives recycling through the clathrin-independent endocytic route.

Previous studies have demonstrated that membrane tubule-mediated transport events in biosynthetic and endocytic routes require phospholipase A2 (PLA2) activity. Here, we show that cytosolic phospholipase A2ε (cPLA2ε, also known as PLA2G4E) is targeted to the membrane compartments of the clathrin-independent endocytic route through a C-terminal stretch of positively charged amino acids, which allows the enzyme to interact with phosphoinositide lipids [especially PI(4,5)P2] that are enriched in clathrin-independent endosomes. Ablation of cPLA2ε suppressed the formation of tubular elements that carry internalized clathrin-independent cargoes, such as MHC-I, CD147 and CD55, back to the cell surface and, therefore, caused their intracellular retention. The ability of cPLA2ε to support recycling through tubule formation relies on the catalytic activity of the enzyme, because the inactive cPLA2ε(S420A) mutant was not able to recover either tubule growth or transport from clathrin-independent endosomes. Taken together, our findings indicate that cPLA2ε is a new important regulator of trafficking processes within the clathrin-independent endocytic and recycling route. The affinity of cPLA2ε for this pathway supports a new hypothesis that different PLA2 enzymes use selective targeting mechanisms to regulate tubule formation locally during specific trafficking steps in the secretory and/or endocytic systems.

Phospholipase A2IVα regulates phagocytosis independent of its enzymatic activity.

Group IVα phospholipase A(2) (PLA(2)IVα) is a lipolytic enzyme that catalyzes the hydrolysis of membrane phospholipids to generate precursors of potent inflammatory lipid mediators. Here, the role of PLA(2)IVα in Fc receptor (FcR)-mediated phagocytosis was investigated, demonstrating that PLA(2)IVα is selectively activated upon FcR-mediated phagocytosis in macrophages and that it rapidly translocates to the site of the nascent phagosome. Moreover, pharmacological inhibition of PLA(2)IVα by pyrrophenone reduces particle internalization by up to 50%. In parallel, fibroblasts from PLA(2)IVα knock-out mice overexpressing FcγRIIA and able to internalize IgG-opsonized beads show 50% lower phagocytosis, compared with wild-type cells, and transfection of PLA(2)IVα fully recovers this impaired function. Interestingly, transfection of the catalytically inactive deleted PLA(2)IVα mutant (PLA(2)IVα(1-525)) and point mutant (PLA(2)IVα-S228C) also promotes recovery of this impaired function. Finally, transfection of the PLA(2)IVα C2 domain (which is directly involved in PLA(2)IVα membrane binding), but not of PLA(2)IVα-D43N (which cannot bind to membranes), rescues FcR-mediated phagocytosis. These data unveil a new mechanism of action for PLA(2)IVα, which demonstrates that the membrane binding, and not the enzymatic activity, is required for PLA(2)IVα modulation of FcR-mediated phagocytosis.

The developmentally regulated osteoblast phosphodiesterase GDE3 is glycerophosphoinositol-specific and modulates cell growth.

The glycerophosphodiester phosphodiesterase enzyme family involved in the hydrolysis of glycerophosphodiesters has been characterized in bacteria and recently identified in mammals. Here, we have characterized the activity and function of GDE3, one of the seven mammalian enzymes. GDE3 is up-regulated during osteoblast differentiation and can affect cell morphology. We show that GDE3 is a glycerophosphoinositol (GroPIns) phosphodiesterase that hydrolyzes GroPIns, producing inositol 1-phosphate and glycerol, and thus suggesting specific roles for this enzyme in GroPIns metabolism. Substrate specificity analyses show that wild-type GDE3 selectively hydrolyzes GroPIns over glycerophosphocholine, glycerophosphoethanolamine, and glycerophosphoserine. A single point mutation in the catalytic domain of GDE3 (GDE3R231A) leads to loss of GroPIns enzymatic hydrolysis, identifying an arginine residue crucial for GDE3 activity. After heterologous GDE3 expression in HEK293T cells, phosphodiesterase activity is detected in the extracellular medium, with no effect on the intracellular GroPIns pool. Together with the millimolar concentrations of calcium required for GDE3 activity, this predicts an enzyme topology with an extracellular catalytic domain. Interestingly, GDE3 ectocellular activity is detected in a stable clone from a murine osteoblast cell line, further confirming the activity of GDE3 in a more physiological context. Finally, overexpression of wild-type GDE3 in osteoblasts promotes disassembly of actin stress fibers, decrease in growth rate, and increase in alkaline phosphatase activity and calcium content, indicating a role for GDE3 in induction of differentiation. Thus, we have identified the GDE3 substrate GroPIns as a candidate mediator for osteoblast proliferation, in line with the GroPIns activity observed previously in epithelial cells.

Cytosolic phospholipase A2 alpha regulates cell growth in RET/PTC-transformed thyroid cells.

Modulation of cytosolic phospholipase A(2) (PLA(2)) expression levels and production of its metabolites have been reported in several tumor types, indicating involvement of arachidonic acid and its derivatives in tumorigenesis. Following our demonstration that the PLA(2) group IV isoform alpha (PLA(2)IV alpha) controls TSH-independent growth of normal thyroid (PCCl(3)) cells, we have investigated the mitogenic role of PLA(2)IV alpha in rat thyroid cells transformed by the RET/PTC oncogenes (PC-PTC cells). We now report that PLA(2)IV alpha acts downstream of the RET/PTC oncogenes in a novel pathway controlling RET-dependent cell proliferation. In addition, we show that PLA(2)IV alpha is in its phosphorylated/active form not only in RET/PTC-transformed cells and in cells derived from human papillary carcinomas but also in lysates from tumor tissues, thus relating constitutive activation of PLA(2)IV alpha to RET/PTC-dependent tumorigenesis. Moreover, p38 stress-activated protein kinase is the downstream effector of RET/PTC that is responsible for PLA(2)IV alpha phosphorylation and activity. In summary, our data elucidate a novel mechanism in the control of thyroid tumor cell growth that is induced by the RET/PTC oncogenes and which is distinguishable from that of other oncogenes, such as BRAF. This mechanism is mediated by PLA(2)IV alpha and should be amenable to targeted pharmacologic intervention.

Analysis of phosphoinositides and their aqueous metabolites.

Many and various experimental techniques have been developed to fully analyze the intracellular signaling pathways of membrane phosphoinositides and their water-soluble derivatives. This chapter concentrates mainly on the range of lipid-derived, water-soluble signaling molecules that can be produced in cells from these membrane phosphoinositides, for which we and others have proposed biological roles. These include lysophosphatidylinositol, produced via phospholipase A(1/2) activities on phosphatidylinositol; cyclic inositol phosphates, produced via phosphatidylinositol/lysophosphatidylinositol-specific phospholipase C activities; and glycerophosphoinositols, produced via lysophospholipase A(2/1) activities on their corresponding lysophosphoinositides. While the methodologies described in this chapter are aimed more specifically at the separation, identification, and quantification of monophosphorylated glyceropho sphoinositols and other similarly charged inositol-containing products of the membrane phosphoinositides in cell extracts, they can be equally well applied to the full range of lysophosphoinositides, glycerophosphoinositols, inositol phosphates, and further inositol-containing water-soluble products of the phosphoinositides (e.g., cyclic inositol phosphates, methylphosphoinositol phosphates).

Molecular characterization of a glycerophosphoinositol transporter in mammalian cells.

The glycerophosphoinositols are ubiquitous phosphoinositide metabolites involved in the control of several cell functions. They exert their actions both intracellularly and by rapidly equilibrating across the plasma membrane when added to cells, implying the existence of a transporter for their membrane permeation. Such a transporter, GIT1, has been cloned in yeast. By PSI-BLAST analysis, we have identified the Glut2 transporter as a human-genome candidate ortholog of GIT1. This was supported directly through the use of inhibitors, siRNAs and competition studies of specific uptake of GroPIns in HeLa cells over-expressing human Glut2. These data identify Glut2 as a GroPIns transporter in mammals, and define a physiologically relevant cell-permeation mechanism.

A novel pathway of cell growth regulation mediated by a PLA2alpha-derived phosphoinositide metabolite.

The phosphoinositides have well-defined roles in the control of cellular functions, including cytoskeleton dynamics, membrane trafficking, and cell signaling. However, the interplay among the phosphoinositides and their diffusible derivatives that originate through phospholipase A2 action (the lysophosphoinositides and glycerophosphoinositols) remains to be fully elucidated. Here we demonstrate that in PCCl3 rat thyroid cells, the intracellular levels of glycerophosphoinositol are finely modulated by ATP and norepinephrine through the P2Y metabotropic and alpha-adrenergic receptors, respectively. The enzyme involved here is phospholipase A2 IValpha (PLA2 IValpha), which in these cells specifically hydrolyzes phosphatidylinositol, forming lysophosphatidylinositol, glycerophosphoinositol, and arachidonic acid. This receptor-mediated activation of PLA2 IValpha leads to stimulation of PCCl3 cell growth. The involvement of a PLA2 IValpha-mediated pathway is demonstrated by inhibition of the increase in intracellular glycerophosphoinositol levels and cell proliferation by specific inhibitors, RNA interference, and overexpression of the dominant-negative PLA2 IValpha(1-522). Modulation of PCCl3 cell growth is not seen with inhibitors of arachidonic acid metabolism. In conclusion, these data characterize glycerophosphoinositol as a mediator of the purinergic and adrenergic regulation of PCCl3 cell proliferation, defining a novel regulatory cascade specifically involving this soluble phosphoinositide derivative and widening the involvement of the phosphoinositides in the regulation of cell function.

Reorganization of actin cytoskeleton by the phosphoinositide metabolite glycerophosphoinositol 4-phosphate.

Glycerophosphoinositol 4-phosphate (GroPIns-4P) is a biologically active, water-soluble phospholipase A metabolite derived from phosphatidylinositol 4-phosphate, whose cellular concentrations have been reported to increase in Ras-transformed cells. It is therefore important to understand its biological activities. Herein, we have examined whether GroPIns-4P can regulate the organization of the actin cytoskeleton, because this could be a Ras-related function involved in cell motility and metastatic invasion. We find that in serum-starved Swiss 3T3 cells, exogenously added GroPIns-4P rapidly and potently induces the formation of membrane ruffles, and, later, the formation of stress fibers. These actin structures can be regulated by the small GTPases Cdc42, Rac, and Rho. To analyze the mechanism of action of GroPIns-4P, we selectively inactivated each of these GTPases. GroPIns-4P requires active Rac and Rho, but not Cdc42, for ruffle and stress fiber formation, respectively. Moreover, GroPIns-4P induces a rapid translocation of the green fluorescent protein-tagged Rac into ruffles, and increases the fraction of GTP-bound Rac, in intact cells. The activation of Rac by GroPIns-4P was near maximal and long-lasting. Interestingly, this feature seems to be critical in the induction of actin ruffles by GroPIns-4P.

Biological activities and metabolism of the lysophosphoinositides and glycerophosphoinositols.

The lysophospholipids are integral components of the plasma membrane that have often been considered as side products of the phospholipase A2 (PLA2)-dependent production of arachidonic acid and the deacylation/reacylation processes involved in phospholipid homeostasis. Data indicating roles of these lipid derivatives in hormone responses and cell transformation have now led to a different view, and the understanding of their involvement in the modulation of cell function is building up. Here, we will summarise the current knowledge concerning the biological roles of the lysophosphoinositides and the glycerophosphoinositols (their fully deacylated counterparts) in the framework of their known effects, and those of the other lysophospholipids and glycerophospholipids.