Phagocytosis of non-opsonized microorganisms by macrophages initiates innate immune responses for host defense against infection. Cytosolic phospholipase A 2 is activated during phagocytosis, releasing arachidonic acid for production of eicosanoids, which initiate acute inflammation. Our objective was to identify pattern recognition receptors that stimulate arachidonic acid release and cyclooxygenase 2 (COX2) expression in macrophages by pathogenic yeast and yeast cell walls. Zymosan-and Candida albicans-stimulated arachidonic acid release from resident mouse peritoneal macrophages was blocked by soluble glucan phosphate. In RAW264.7 cells arachidonic acid release, COX2 expression, and prostaglandin production were enhanced by overexpressing the ␤-glucan receptor, dectin-1, but not dectin-1 lacking the cytoplasmic tail. Pure particulate (1, 3)-␤-D-glucan stimulated arachidonic acid release and COX2 expression, which were augmented in a Tolllike receptor 2 (TLR2)-dependent manner by macrophage-activating lipopeptide-2. However, arachidonic acid release and leukotriene C 4 production stimulated by zymosan and C. albicans were TLR2-independent, whereas COX2 expression and prostaglandin production were partially blunted in TLR2

Group IVA cytosolic phospholipase A 2 (cPLA 2 ␣) is regulated by phosphorylation and calcium-induced translocation to membranes. Immortalized mouse lung fibroblasts lacking endogenous cPLA 2 ␣ (IMLF ؊/؊ ) were reconstituted with wild type and cPLA 2 ␣ mutants to investigate how calcium, phosphorylation, and the putative phosphatidylinositol 4,5-bisphosphate (PIP 2 ) binding site regulate translocation and arachidonic acid (AA) release. Agonists that elicit distinct modes of calcium mobilization were used. Serum induced cPLA 2 ␣ translocation to Golgi within seconds that temporally paralleled the initial calcium transient. However, the subsequent influx of extracellular calcium was essential for stable binding of cPLA 2 ␣ to Golgi and AA release. In contrast, phorbol 12-myristate 13-acetate induced low amplitude calcium oscillations, slower translocation of cPLA 2 ␣ to Golgi, and much less AA release, which were blocked by chelating extracellular calcium. AA release from IMLF ؊/؊ expressing phosphorylation site (S505A) and PIP 2 binding site (K488N/K543N/K544N) mutants was partially reduced compared with cells expressing wild type cPLA 2 ␣, but calcium-induced translocation was not impaired. Consistent with these results, Ser-505 phosphorylation did not change the calcium requirement for interfacial binding and catalysis in vitro but increased activity by 2-fold. Mutations in basic residues in the catalytic domain of cPLA 2 ␣ reduced activation by PIP 2 but did not affect the concentration of calcium required for interfacial binding or phospholipid hydrolysis. The results demonstrate that Ser-505 phosphorylation and basic residues in the catalytic domain principally act to regulate cPLA 2 ␣ hydrolytic activity.

No abstract

No abstract

In this study, we identify the principal splice variant of human cytosolic phospholipase A 2 ␤ (cPLA 2 ␤) (also known as Group IVB cPLA 2 ) present in cells. In human lung, spleen, and ovary and in a lung epithelial cell line (BEAS-2B), cPLA 2 ␤ is expressed as a 100-kDa protein, not the 114-kDa form originally predicted. Using RNA interference, the 100-kDa protein in BEAS-2B cells was confirmed to be cPLA 2 ␤. BEAS-2B cells contain three different RNA splice variants of cPLA 2 ␤ (␤1, ␤2, and ␤3). cPLA 2 ␤1 is identical to the previously cloned cPLA 2 ␤, predicted to encode a 114-kDa protein. However, cPLA 2 ␤2 and cPLA 2 ␤3 splice variants are smaller and contain internal deletions in the catalytic domain. The 100-kDa cPLA 2 ␤ in BEAS-2B cells is the translated product of cPLA 2 ␤3. cPLA 2 ␤3 exhibits calcium-dependent PLA 2 activity against palmitoyl-arachidonyl-phosphatidylethanolamine and low level lysophospholipase activity but no activity against phosphatidylcholine. Unlike Group IVA cPLA 2 ␣, cPLA 2 ␤3 is constitutively bound to membrane in unstimulated cells, localizing to mitochondria and early endosomes. cPLA 2 ␤3 is widely expressed in tissues, suggesting that it has a generalized function at these unique sites.Phospholipase A 2 (PLA 2 ) 2 enzymes catalyze hydrolysis of sn-2 acyl chains from membrane phospholipids. They execute diverse functions, such as digestion of dietary phospholipids, microbial degradation, membrane remodeling, and production of lipid mediators. Traditionally, PLA 2 s are grouped depending on their active site residues, requirements for calcium, and localization in the cell. Three main classes of PLA 2 s are Group VI intracellular calcium-independent PLA 2 s, low molecular weight secreted PLA 2 s, and Group IV cytosolic PLA 2 s (1, 2). Group IVA cytosolic PLA 2 ␣ has received special attention, because it is the only PLA 2 that selectively hydrolyzes arachidonic acid from the sn-2 position of membrane phospholipids (1). Arachidonic acid is the precursor of prostaglandins and leukotrienes (1, 3). Mice genetically deficient in cPLA 2 ␣ have provided evidence for its critical role in regulating physiological processes and various diseases (4 -11). cPLA 2 ␣ contains an N-terminal calcium binding domain (C2 domain) and a C-terminal catalytic domain (12). Calcium binds to the C2 domain and facilitates the translocation of the enzyme from cytosol to the Golgi, endoplasmic reticulum, and nuclear envelope (13-16). Five other members of the Group IV cPLA 2 family, cPLA 2 ␤ (Group IVB), cPLA 2 ␥ (Group IVC), cPLA 2 ␦ (Group IVD), cPLA 2 (Group IVE), and cPLA 2 (Group IVF), have been identified (17-21). cPLA 2 ␦, -, and -are clustered near cPLA 2 ␤ on mouse chromosome 2 and have more homology to cPLA 2 ␤ than to cPLA 2 ␣ or cPLA 2 ␥ (21). From analysis of the human genome, cPLA 2 ␤ is similarly positioned near cPLA 2 ␦, -, and -on chromosome 15. All members of the Group IV family have a conserved Ser/Asp dyad necessary for catalysis (12).Human cPLA 2 ␤ was originally cloned from human brain ...