Phospholipase A2 (PLA2) enzymes are critical regulators of prostaglandin and leukotriene synthesis and can directly modify the composition of cellular membranes. PLA2 enzymes release fatty acids and lysophospholipids, including the precursor of platelet-activating factor, PAF, from phospholipids. Free fatty acids, eicosanoids, lysophospholipids and PAF are potent regulators of inflammation, reproduction and neurotoxicity. The physiological roles of the various forms of PLA2 are not well defined. The cytosolic form, cPLA2, preferentially releases arachidonic acid from phospholipids and is regulated by changes in intracellular calcium concentration. We have now created 'knockout' (cPLA2-/-) mice that lack this enzyme, in order to evaluate its physiological importance. We find that cPLA2-/- mice develop normally, but that the females produce only small litters in which the pups are usually dead. Stimulated peritoneal macrophages from cPLA2-/- animals did not produce prostaglandin E2 or leukotriene B4 or C4. After transient middle cerebral artery occlusion, cPLA2-/- mice had smaller infarcts and developed less brain oedema and fewer neurological deficits. Thus cPLA2 is important for macrophage production of inflammatory mediators, fertility, and in the pathophysiology of neuronal death after transient focal cerebral ischaemia.

The Krüppel-associated box A (KRAB-A) domain is an evolutionarily conserved transcriptional repressor domain present in approximately one-third of zinc finger proteins of the Cys 2 -His 2 type. Using the yeast two-hybrid system, we report the isolation of a cDNA encoding a novel murine protein, KRAB-A interacting protein 1 (KRIP-1) that physically interacts with the KRAB-A region. KRIP-1 is a member of the RBCC subfamily of the RING finger, or Cys 3 HisCys 4 , family of zinc binding proteins whose other members are known to play important roles in differentiation, oncogenesis, and signal transduction. The KRIP-1 protein has high homology to TIF1, a putative modulator of liganddependent activation function of nuclear receptors. A 3.5-kb mRNA for KRIP-1 is ubiquitously expressed among all adult mouse tissues studied. When a GAL4-KRIP-1 fusion protein is expressed in COS cells with a chloramphenicol acetyltransferase reporter construct with five GAL4 binding sites, there is dose-dependent repression of transcription. Thus, KRIP-1 interacts with the KRAB-A region of C 2 H 2 zinc finger proteins and may mediate or modulate KRAB-A transcriptional repressor activity.

In this study, we determined the functional significance and mechanism of the exogenous hVPLA 2 -induced arachidonic acid (AA) release and leukotriene C 4 (LTC 4 ) synthesis in isolated human peripheral blood eosinophils. As low a concentration as 10 nM exogenous hVPLA 2 was able to elicit the significant release of AA and LTC 4 from unstimulated eosinophils, which depended on its ability to act on phosphatidylcholine membranes. hVPLA 2 also augmented the release of AA and LTC 4 from eosinophils activated with formyl-Met-Leu-Phe ؉ cytochalasin B. A cellular fluorescent PLA 2 assay showed that hVPLA 2 had a lipolytic action first on the outer plasma membrane and then on the perinuclear region. hVPLA 2 also caused the translocation of 5-lipoxygenase from the cytosol to the nuclear membrane and a 2-fold increase in 5-lipoxygenase activity. However, hVPLA 2 induced neither the increase in intracellular calcium concentration nor cPLA 2 phosphorylation; consequently, cPLA 2 activity was not affected by hVPLA 2 . Pharmacological inhibition of cPLA 2 and the hVPLA 2 -induced activation of eosinophils derived from the cPLA 2 -deficient mouse corroborated that hVPLA 2 mediates the release of AA and leukotriene in a cPLA 2 -independent manner. As such, this study represents a unique example in which a secretory phospholipase induces the eicosanoid formation in inflammatory cells, completely independent of cPLA 2 activation.