Eileen O'Leary

Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2

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.

Deletion of cytosolic phospholipase A(2) suppresses Apc(Min)-induced tumorigenesis.

Although nonsteroidal antiinflammatory drugs (NSAIDs) show great promise as therapies for colon cancer, a dispute remains regarding their mechanism of action. NSAIDs are known to inhibit cyclooxygenase (COX) enzymes, which convert arachidonic acid (AA) to prostaglandins (PGs). Therefore, NSAIDs may suppress tumorigenesis by inhibiting PG synthesis. However, various experimental studies have suggested the possibility of PG-independent mechanisms. Notably, disruption of the mouse group IIA secretory phospholipase A2 locus (Pla2g2a), a potential source of AA for COX-2, increases tumor number despite the fact that the mutation has been predicted to decrease PG production. Some authors have attempted to reconcile the results by suggesting that the level of the precursor (AA), not the products (PGs), is the critical factor. To clarify the role of AA in tumorigenesis, we have examined the effect of deleting the group IV cytosolic phospholipase A2 (cPLA2) locus (Pla2g4). We report that ApcMin/+, cPLA2−/− mice show an 83% reduction in tumor number in the small intestine compared with littermates with genotypes ApcMin/+, cPLA2+/− and ApcMin/+, cPLA2+/+. This tumor phenotype parallels that of COX-2 knockout mice, suggesting that cPLA2 is the predominant source of AA for COX-2 in the intestine. The protective effect of cPLA2 deletion is thus most likely attributed to a decrease in the AA supply to COX-2 and a resultant decrease in PG synthesis. The tumorigenic effect of sPLA2 mutations is likely to be through a completely different pathway.

Kid-1, a putative renal transcription factor: regulation during ontogeny and in response to ischemia and toxic injury.

We have identified a new putative transcription factor from the rat kidney, termed Kid-1 (for kidney, ischemia and developmentally regulated gene 1). Kid-1 belongs to the C2H2 class of zinc finger genes. Its mRNA accumulates with age in postnatal renal development and is detected predominantly in the kidney. Kid-1 mRNA levels decline after renal injury secondary to ischemia or folic acid administration, two insults which result in epithelial cell dedifferentiation, followed by regenerative hyperplasia and differentiation. The low expression of Kid-1 early in postnatal development, and when renal tissue is recovering after injury, suggests that the gene product is involved in establishment of a differentiated phenotype and/or regulation of the proliferative response. The deduced protein contains 13 C2H2 zinc fingers at the COOH end in groups of 4 and 9 separated by a 32-amino-acid spacer. There are consensus sites for phosphorylation in the NH2 terminus non-zinc finger region as well as in the spacer region between zinc fingers 4 and 5. A region of the deduced protein shares extensive homology with a catalytic region of Raf kinases, a feature shared only with TFIIE among transcription factors. To determine whether Kid-1 can modulate transcription, a chimeric construct encoding the Kid-1 non-zinc finger region (sense or antisense) and the DNA-binding region of GAL4 was transfected into COS and LLC-PK1 cells together with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 binding sites, driven by either a minimal promoter or a simian virus 40 enhancer. CAT activity was markedly inhibited in cells transfected with the sense construct compared with the activity in cells transfected with the antisense construct. To our knowledge, this pattern of developmental regulation, kidney expression, and regulation of transcription is unique among the C2H2 class of zinc finger-containing DNA-binding proteins.

TRIP-Br: a novel family of PHD zinc finger- and bromodomain-interacting proteins that regulate the transcriptional activity of E2F-1/DP-1

We report the isolation of TRIP‐Br1, a transcriptional regulator that interacts with the PHD‐bromodomain of co‐repressors of Krüppel‐associated box (KRAB)‐mediated repression, KRIP‐1(TIF1β) and TIF1α, as well as the co‐activator/adaptor p300/CBP. TRIP‐Br1 and the related protein TRIP‐Br2 possess transactivation domains. Like MDM2, which has a homologous transactivation domain, TRIP‐Br proteins functionally contact DP‐1, stimulating E2F‐1/DP‐1 transcriptional activity. KRIP‐1 potentiates TRIP‐Br protein co‐activation of E2F‐1/DP‐1. TRIP‐Br1 is a component of a multiprotein complex containing E2F‐1 and DP‐1. Co‐expression of the retinoblastoma gene product (RB) abolishes baseline E2F‐1/DP‐1 transcriptional activity as well as TRIP‐Br/KRIP‐1 co‐activation, both of which are restored by the adenovirus E1A oncoprotein. These features suggest that TRIP‐Br proteins function at E2F‐responsive promoters to integrate signals provided by PHD‐ and/or bromodomain‐ containing transcription factors. TRIP‐Br1 is identical to the cyclin‐dependent kinase 4 (cdk4)‐binding protein p34SEI‐1, which renders the activity of cyclin D/cdk4 resistant to the inhibitory effect of p16INK4a during late G1. TRIP‐Br1(p34SEI‐1) is differentially overexpressed during the G1 and S phases of the cell cycle, consistent with a dual role for TRIP‐Br1(p34SEI‐1) in the regulation of cell cycle progression through sequential effects on the transcriptional activity of E2F‐responsive promoters during G1 and S phases.

Human Group V Phospholipase A2 Induces Group IVA Phospholipase A2-independent Cysteinyl Leukotriene Synthesis in Human Eosinophils

We previously reported that exogenously added human group V phospholipase A2 (hVPLA2) could elicit leukotriene B4 biosynthesis in human neutrophils through the activation of group IVA phospholipase A2 (cPLA2) (Kim, Y. J., Kim, K. P., Han, S. K., Munoz, N. M., Zhu, X., Sano, H., Leff, A. R., and Cho, W. (2002) J. Biol. Chem. 277, 36479-36488). In this study, we determined the functional significance and mechanism of the exogenous hVPLA2-induced arachidonic acid (AA) release and leukotriene C4 (LTC4) synthesis in isolated human peripheral blood eosinophils. As low a concentration as 10 nm exogenous hVPLA2 was able to elicit the significant release of AA and LTC4 from unstimulated eosinophils, which depended on its ability to act on phosphatidylcholine membranes. hVPLA2 also augmented the release of AA and LTC4 from eosinophils activated with formyl-Met-Leu-Phe + cytochalasin B. A cellular fluorescent PLA2 assay showed that hVPLA2 had a lipolytic action first on the outer plasma membrane and then on the perinuclear region. hVPLA2 also caused the translocation of 5-lipoxygenase from the cytosol to the nuclear membrane and a 2-fold increase in 5-lipoxygenase activity. However, hVPLA2 induced neither the increase in intracellular calcium concentration nor cPLA2 phosphorylation; consequently, cPLA2 activity was not affected by hVPLA2. Pharmacological inhibition of cPLA2 and the hVPLA2-induced activation of eosinophils derived from the cPLA2-deficient mouse corroborated that hVPLA2 mediates the release of AA and leukotriene in a cPLA2-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 cPLA2 activation.

Deletion of cytosolic phospholipase A2 promotes striated muscle growth.

Generation of arachidonic acid by the ubiquitously expressed cytosolic phospholipase A2 (PLA2) has a fundamental role in the regulation of cellular homeostasis, inflammation and tumorigenesis. Here we report that cytosolic PLA2 is a negative regulator of growth, specifically of striated muscle. We find that normal growth of skeletal muscle, as well as normal and pathologic stress-induced hypertrophic growth of the heart, are exaggerated in Pla2g4a−/− mice, which lack the gene encoding cytosolic PLA2. The mechanism underlying this phenotype is that cytosolic PLA2 negatively regulates insulin-like growth factor (IGF)-1 signaling. Absence of cytosolic PLA2 leads to sustained activation of the IGF-1 pathway, which results from the failure of 3-phosphoinositide-dependent protein kinase (PDK)-1 to recruit and phosphorylate protein kinase C (PKC)-ζ, a negative regulator of IGF-1 signaling. Arachidonic acid restores activation of PKC-ζ, correcting the exaggerated IGF-1 signaling. These results indicate that cytosolic PLA2 and arachidonic acid regulate striated muscle growth by modulating multiple growth-regulatory pathways.

PLIP, a novel splice variant of Tip60, interacts with group IV cytosolic phospholipase A(2), induces apoptosis, and potentiates prostaglandin production.

ABSTRACT The group IV cytosolic phospholipase A2(cPLA2) has been localized to the nucleus (M. R. Sierra-Honigmann, J. R. Bradley, and J. S. Pober, Lab. Investig. 74:684–695, 1996) and is known to translocate from the cytosolic compartment to the nuclear membrane (S. Glover, M. S. de Carvalho, T. Bayburt, M. Jonas, E. Chi, C. C. Leslie, and M. H. Gelb, J. Biol. Chem. 270:15359–15367, 1995; A. R. Schievella, M. K. Regier, W. L. Smith, and L. L. Lin, J. Biol. Chem. 270:30749–30754, 1995). We hypothesized that nuclear proteins interact with cPLA2 and participate in the functional effects of this translocation. We have identified a nuclear protein, cPLA2-interacting protein (PLIP), a splice variant of human Tip60, which interacts with the amino terminal region of cPLA2. Like Tip60, PLIP cDNA includes the MYST domain containing a C2HC zinc finger and well-conserved similarities to acetyltransferases. Both PLIP and Tip60 coimmunoprecipitate and colocalize with cPLA2 within the nuclei of transfected COS cells. A polyclonal antibody raised to PLIP recognizes both PLIP and Tip60. Endogenous Tip60 and/or PLIP in rat mesangial cells is localized to the nucleus in response to serum deprivation. Nuclear localization coincides temporally with apoptosis. PLIP expression, mediated by adenoviral gene transfer, potentiates serum deprivation-induced prostaglandin E2 (PGE2) production and apoptosis in mouse mesangial cells from cPLA2 +/+ mice but not in mesangial cells derived from cPLA2 −/− mice. Thus PLIP, a splice variant of Tip60, interacts with cPLA2 and potentiates cPLA2-mediated PGE2 production and apoptosis.

Expression of the Transcriptional Repressor Protein Kid-1 Leads to the Disintegration of the Nucleolus

The rat Kid-1 gene codes for a 66-kDa protein with KRAB domains at the NH2 terminus and two Cys2His2-zinc finger clusters of four and nine zinc fingers at the COOH terminus. It was the first KRAB-zinc finger protein for which a transcriptional repressor activity was demonstrated. Subsequently, the KRAB-A domain was identified as a widespread transcriptional repressor motif. We now present a biochemical and functional analysis of the Kid-1 protein in transfected cells. The full-length Kid-1 protein is targeted to the nucleolus and adheres tightly to as yet undefined nucleolar structures, leading eventually to the disintegration of the nucleolus. The tight adherence and nucleolar distribution can be attributed to the larger zinc finger cluster, whereas the KRAB-A domain is responsible for the nucleolar fragmentation. Upon disintegration of the nucleolus, the nucleolar transcription factor upstream binding factor disappears from the nucleolar fragments. In the absence of Kid-1, the KRIP-1 protein, which represents the natural interacting partner of zinc finger proteins with a KRAB-A domain, is homogeneously distributed in the nucleus, whereas coexpression of Kid-1 leads to a shift of KRIP-1 into the nucleolus. Nucleolar run-ons demonstrate that rDNA transcription is shut off in the nucleolar fragments. Our data demonstrate the functional diversity of the KRAB and zinc finger domains of Kid-1 and provide new functional insights into the regulation of the nucleolar structure.

Identification of PP2A as a novel interactor and regulator of TRIP-Br1

TRIP-Br proteins are a novel family of transcriptional coregulators involved in E2F-mediated cell cycle progression. Three of the four mammalian members of TRIP-Br family, including TRIP-Br1, are known oncogenes. We now report the identification of the Balpha regulatory subunit of serine/threonine protein phosphatase 2A (PP2A) as a novel TRIP-Br1 interactor, based on an affinity binding assay coupled with mass spectrometry. A GST-TRIP-Br1 fusion protein associates with catalytically active PP2A-ABalphaC holoenzyme in vitro. Coimmunoprecipitation confirms this association in vivo. Immunofluorescence staining with a monoclonal antibody against TRIP-Br1 reveals that endogenous TRIP-Br1 and PP2A-Balpha colocalize mainly in the cytoplasm. Consistently, immunoprecipitation followed by immunodetection with anti-phosphoserine antibody suggest that TRIP-Br1 exists in a serine-phosphorylated form. Inhibition of PP2A activity by okadaic acid or transcriptional silencing of the PP2A catalytic subunit by small interfering RNA results in downregulation of total TRIP-Br1 protein levels but upregulation of serine-phosphorylated TRIP-Br1. Overexpression of PP2A catalytic subunit increases TRIP-Br1 protein levels and TRIP-Br1 co-activated E2F1/DP1 transcription. Our data support a model in which association between PP2A-ABalphaC holoenzyme and TRIP-Br1 in vivo in mammalian cells represents a novel mechanism for regulating the level of TRIP-Br1 protooncoprotein.

TIM2 Gene Deletion Results in Susceptibility to Cisplatin-Induced Kidney Toxicity

T-cell Immunoglobulin and Mucin domain 2 (TIM2) belongs to the receptor family of cell surface molecules expressed on kidney, liver, and T cells. Previous studies have revealed that TIM2-deficient mice (TIM2(-/-)) are more susceptible to the Th2-mediated immune response in an airway inflammation model. Here, we investigated the phenotypic response of TIM2(-/-) mice to cisplatin-induced kidney toxicity. A lethality study in male BALB/c wild-type (TIM2(+/+)) and TIM2(-/-) mice, administered with 20 mg/kg cisplatin ip, resulted in 80% mortality of TIM2(-/-) mice as compared with 30% mortality in the TIM2(+/+) group by day 5. The TIM2(-/-) mice showed approximately fivefold higher injury as estimated by blood urea nitrogen and serum creatinine at 48 h that was confirmed by significantly increased proximal tubular damage assessed histologically (H & E staining). A significantly higher expression of Th2-associated cytokines, TNF-α, IL-1β, IL-6, and TGFβ, with a significant reduction of Th1-associated cytokines, RANTES and MCP-1, by 72 h was observed in the TIM2(-/-) mice as compared with TIM2(+/+) mice. A higher baseline protein expression of caspase-3 (approximately twofold) coupled with an early onset of p53 protein activation by 48 h resulted in an increased apoptosis by 48-72 h in TIM2(-/-) compared with TIM2(+/+). In conclusion, the increased expression of the proinflammatory and proapoptotic genes, with a higher number of apoptotic cells, and a pronounced increase in injury and mortality of the TIM2-deficient mice collectively suggest a protective role of TIM2 in cisplatin-induced nephrotoxicity.