cPLA2α Enzyme Inhibition Attenuates Inflammation and Keratinocyte Proliferation.

As a regulator of cellular inflammation and proliferation, cytosolic phospholipase A2 α (cPLA2α) is a promising therapeutic target for psoriasis; indeed, the cPLA2α inhibitor AVX001 has shown efficacy against plaque psoriasis in a phase I/IIa clinical trial. To improve our understanding of the anti-psoriatic properties of AVX001, we sought to determine how the compound modulates inflammation and keratinocyte hyperproliferation, key characteristics of the psoriatic epidermis. We measured eicosanoid release from human peripheral blood mononuclear cells (PBMC) and immortalized keratinocytes (HaCaT) and studied proliferation in HaCaT grown as monolayers and stratified cultures. We demonstrated that inhibition of cPLA2α using AVX001 produced a balanced reduction of prostaglandins and leukotrienes; significantly limited prostaglandin E2 (PGE2) release from both PBMC and HaCaT in response to pro-inflammatory stimuli; attenuated growth factor-induced arachidonic acid and PGE2 release from HaCaT; and inhibited keratinocyte proliferation in the absence and presence of exogenous growth factors, as well as in stratified cultures. These data suggest that the anti-psoriatic properties of AVX001 could result from a combination of anti-inflammatory and anti-proliferative effects, probably due to reduced local eicosanoid availability.

Critical Involvement of Calcium-Dependent Cytosolic Phospholipase A2α in Aortic Valve Interstitial Cell Calcification.

The involvement of calcium-dependent cytosolic phospholipase A2α (cPLA2α) in aortic valve calcification is not exhaustively elucidated. Here, cPLA2α expression in aortic valve interstitial cell (AVIC) pro-calcific cultures simulating either metastatic or dystrophic calcification was estimated by qPCR, Western blotting, and counting of cPLA2α-immunoreactive cells, with parallel ultrastructural examination of AVIC calcific degeneration. These evaluations also involved pro-calcific AVIC cultures treated with cPLA2α inhibitor dexamethasone. cPLA2α over-expression resulted for both types of pro-calcific AVIC cultures. Compared to controls, enzyme content was found to increase by up to 300% and 186% in metastatic and dystrophic calcification-like cultures, respectively. Increases in mRNA amounts were also observed, although they were not as striking as those in enzyme content. Moreover, cPLA2α increases were time-dependent and strictly associated with mineralization progression. Conversely, drastically lower levels of enzyme content resulted for the pro-calcific AVIC cultures supplemented with dexamethasone. In particular, cPLA2α amounts were found to decrease by almost 88% and 48% in metastatic and dystrophic calcification-like cultures, respectively, with mRNA amounts showing a similar trend. Interestingly, these drastic decreases in cPLA2α amounts were paralleled by drastic decreases in mineralization degrees, as revealed ultrastructurally. In conclusion, cPLA2α may be regarded as a crucial co-factor contributing to AVIC mineralization in vitro, thus being an attractive potential target for designing novel therapeutic strategies aimed to counteract onset or progression of calcific aortic valve diseases.

Respective contribution of cytosolic phospholipase A2α and secreted phospholipase A2 IIA to inflammation and eicosanoid production in arthritis.

Phospholipase A2s (PLA2) play a key role in generation of eicosanoids. Cytosolic PLA2α (cPLA2α) is constitutively expressed in most cells, whereas IIA secreted PLA2 (sPLA2-IIA) is induced during inflammation and is present at high levels in the synovial fluid of rheumatoid arthritis patients. In mice, both cPLA2α and sPLA2-IIA have been implicated in autoimmune arthritis; however, the respective contribution of these two enzymes to the pathogenesis and production of eicosanoids is unknown. We evaluated the respective role of cPLA2α and sPLA2-IIA with regard to arthritis and eicosanoid profile in an in vivo model of arthritis. While arthritis was most severe in mice expressing both enzymes, it was abolished when both cPLA2α and sPLA2-IIA were lacking. cPLA2α played a dominant role in the severity of arthritis, although sPLA2-IIA sufficed to significantly contribute to the disease. Several eicosanoids were modulated during the course of arthritis and numerous species involved sPLA2-IIA expression. This study confirms the critical role of PLA2s in arthritis and unveils the distinct contribution of cPLA2α and sPLA2-IIA to the eicosanoid profile in arthritis.

Cytosolic Phospholipase A2α Promotes Pulmonary Inflammation and Systemic Disease during Streptococcus pneumoniae Infection.

Pulmonary infection by Streptococcus pneumoniae is characterized by a robust alveolar infiltration of neutrophils (polymorphonuclear cells [PMNs]) that can promote systemic spread of the infection if not resolved. We previously showed that 12-lipoxygenase (12-LOX), which is required to generate the PMN chemoattractant hepoxilin A3 (HXA3) from arachidonic acid (AA), promotes acute pulmonary inflammation and systemic infection after lung challenge with S. pneumoniae As phospholipase A2 (PLA2) promotes the release of AA, we investigated the role of PLA2 in local and systemic disease during S. pneumoniae infection. The group IVA cytosolic isoform of PLA2 (cPLA2α) was activated upon S. pneumoniae infection of cultured lung epithelial cells and was critical for AA release from membrane phospholipids. Pharmacological inhibition of this enzyme blocked S. pneumoniae-induced PMN transepithelial migration in vitro Genetic ablation of the cPLA2 isoform cPLA2α dramatically reduced lung inflammation in mice upon high-dose pulmonary challenge with S. pneumoniae The cPLA2α-deficient mice also suffered no bacteremia and survived a pulmonary challenge that was lethal to wild-type mice. Our data suggest that cPLA2α plays a crucial role in eliciting pulmonary inflammation during pneumococcal infection and is required for lethal systemic infection following S. pneumoniae lung challenge.

Modulation of C-reactive protein and plasma omega-6 fatty acid levels by phospholipase A2 gene polymorphisms following a 6-week supplementation with fish oil.

This clinical trial investigated the impact of a six-week supplementation with fish oil and single nucleotide polymorphisms (SNPs) in PLA2G4A and PLA2G6 genes on total omega-6 fatty acid (n-6 FA) levels in plasma phospholipids (PL) and plasma C-reactive protein (CRP) levels in 191 subjects. Interaction effects between SNPs and supplementation modulated total n-6 FAs and CRP levels in both men and women. Associations between SNPs and total n-6 FA levels and between SNPs and CRP levels were identified in men, independently of supplementation. Supplementation decreased total n-6 FAs without affecting plasma CRP levels. Changes in CRP levels correlated positively with changes in total n-6 FAs in men (r=0.25 p=0.01), but not in women. In conclusion, total n-6 FA levels in plasma PL and plasma CRP levels are modulated by SNPs within PLA2G4A and PLA2G6 genes alone or in combination with fish oil supplementation.

Fever Is Mediated by Conversion of Endocannabinoid 2-Arachidonoylglycerol to Prostaglandin E2.

Fever is a common response to inflammation and infection. The mechanism involves prostaglandin E2 (PGE2)-EP3 receptor signaling in the hypothalamus, which raises the set point of hypothalamic thermostat for body temperature, but the lipid metabolic pathway for pyretic PGE2 production remains unknown. To reveal the molecular basis of fever initiation, we examined lipopolysaccharides (LPS)-induced fever model in monoacylglycerol lipase (MGL)-deficient (Mgll-/-) mice, CB1 receptor-MGL compound-deficient (Cnr1-/-Mgll-/-) mice, cytosolic phospholipase A2α (cPLA2α)-deficient (Pla2g4a-/-) mice, and diacylglycerol lipase α (DGLα)-deficient (Dagla-/-) mice. Febrile reactions were abolished in Mgll-/- and Cnr1-/-Mgll-/- mice, whereas Cnr1-/-Mgll+/+, Pla2g4a-/- and Dagla-/- mice responded normally, demonstrating that MGL is a critical enzyme for fever, which functions independently of endocannabinoid signals. Intracerebroventricular administration of PGE2 caused fever similarly in Mgll-/- and wild-type control mice, suggesting a lack of pyretic PGE2 production in Mgll-/- hypothalamus, which was confirmed by lipidomics analysis. Normal blood cytokine responses after LPS administration suggested that MGL-deficiency does not affect pyretic cytokine productions. Diurnal body temperature profiles were normal in Mgll-/- mice, demonstrating that MGL is unrelated to physiological thermoregulation. In conclusion, MGL-dependent hydrolysis of endocannabinoid 2-arachidonoylglycerol is necessary for pyretic PGE2 production in the hypothalamus.

Promoter RNA links transcriptional regulation of inflammatory pathway genes.

Although many long non-coding RNAs (lncRNAs) have been discovered, their function and their association with RNAi factors in the nucleus have remained obscure. Here, we identify RNA transcripts that overlap the cyclooxygenase-2 (COX-2) promoter and contain two adjacent binding sites for an endogenous miRNA, miR-589. We find that miR-589 binds the promoter RNA and activates COX-2 transcription. In addition to miR-589, fully complementary duplex RNAs that target the COX-2 promoter transcript activate COX-2 transcription. Activation by small RNA requires RNAi factors argonaute-2 (AGO2) and GW182, but does not require AGO2-mediated cleavage of the promoter RNA. Instead, the promoter RNA functions as a scaffold. Binding of AGO2 protein/small RNA complexes to the promoter RNA triggers gene activation. Gene looping allows interactions between the promoters of COX-2 and phospholipase A2 (PLA2G4A), an adjacent pro-inflammatory pathway gene that produces arachidonic acid, the substrate for COX-2 protein. miR-589 and fully complementary small RNAs regulate both COX-2 and PLA2G4A gene expression, revealing an unexpected connection between key steps of the eicosanoid signaling pathway. The work demonstrates the potential for RNA to coordinate locus-dependent assembly of related genes to form functional operons through cis-looping.

Artocarpin attenuates ultraviolet B-induced skin damage in hairless mice by antioxidant and anti-inflammatory effect.

Artocarpin, a prenylated flavonoid isolated from an agricultural plant Artocarpus communis, has been documented to possess anti-inflammation and anticancer activities. As oxidative stress and inflammation promote the development of ultraviolet B (UVB) irradiation-induced photodamage, the aim of the present study was to evaluate the photoprotective effect of artocarpin on UVB-induced skin damage in hairless mice. Artocarpin at a topical dose of 0.05% and 0.1% showed a significant photoprotective effect by decreasing histopathological changes, such as desquamation, epidermal thicken and sunburn cell formation, but 0.1% of artocarpin administration did not show better effect. Regarding the antioxidant activities, artocarpin exhibited a significant effect (P<0.05) by decreasing levels of reactive species oxygen and lipid peroxidation. In addition, artocarpin can significant decrease the level of tumor necrosis factor-α and interleukin-1ß for downregulating the inflammation protein, including the synthesis of cytosolic phospholipase A2 and cyclooxygenase-2 (P<0.05). In conclusion, these data suggest that artocarpin can prevent skin damage from UVB irradiation-induced photodamage in hairless mice and this is likely mediated through its antioxidant and anti-inflammation mechanisms. Therefore, we suggested that artocarpin could be a useful photoprotective agent in medicine and/or cosmetics.

PLA2G4A mutants modified protective effect of tea consumption against colorectal cancer.

PURPOSE: The primary aim was to respectively evaluate PLA2G4A mutants modifying protective effect of tea consumption against colorectal cancer (CRC), colon and rectal cancer. METHODS: All participants were recruited from January 2006 to April 2008. The information about tea consumption was collected by a structured questionnaire. CRC patients were diagnosed based on histology. Four single-nuclear polymorphisms (SNPs) in PLA2G4A gene were selected. Multiple logistic regression models were used for assessing the joint effects between tea consumption and SNPs on CRC, colon and rectal cancer. RESULTS: Three hundred patients with CRC and 296 controls well-matched were used in the final analyses. The significant individual associations between four SNPs (rs6666834, rs10911933, rs4650708 and rs7526089) and CRC were not observed. However, their CTAC haplotype was significantly associated with the increased risk of CRC (OR = 3.06; 95%CI = 1.52-6.19), compared with TCAC haplotype. Drinking tea was correlated with a decreased risk of CRC after adjustment for covariates (OR = 0.61; 95%CI = 0.39-0.97). Meanwhile, compared with no-tea drinkers with TT/CT genotype of rs6666834, tea drinkers with TT/CT or CC had significant lower risk of CRC (OR = 0.6, 95%CI = 0.36-1.00 for TT/CT; 0.38, 0.19-0.74 for CC). The joint effects between the remaining three SNPs and drinking tea on CRC were observed as well. Similar findings were observed on colon and rectal cancers. CONCLUSIONS: Tea consumption and haplotype of mutants in PLA2G4A gene were respectively associated with the risk of CRC. PLA2G4A mutants modified the protective effect of tea consumption against CRC, colon and rectal cancers in Chinese population.

The enteropathy of prostaglandin deficiency.

BACKGROUND: Small intestinal ulcers are frequent complications of therapy with nonsteroidal anti-inflammatory drugs (NSAIDs). We present here a genetic deficiency of eicosanoid biosynthesis that illuminates the mechanism of NSAID-induced ulcers of the small intestine. METHODS: Eicosanoids and metabolites were measured by isotope dilution with mass spectrometry. cDNA was obtained by reverse transcription and sequenced following amplification with RT-PCR. RESULTS: We investigated the cause of chronic recurrent small intestinal ulcers, small bowel perforations, and gastrointestinal blood loss in a 45-year-old man who was not taking any cyclooxygenase inhibitor. Prostaglandin metabolites in urine were significantly depressed. Serum thromboxane B2 (TxB2) production was 4.6% of normal controls (P<0.006), and serum 12-HETE was 1.3% of controls (P<0.005). Optical platelet aggregation with simultaneous monitoring of ATP release demonstrated absent granule secretion in response to ADP and a blunted aggregation response to ADP and collagen, but normal response to arachidonic acid (AA). LTB4 biosynthesis by ionophore-activated leukocytes was only 3% of controls, and urinary LTE4 was undetectable. These findings suggested deficient AA release from membrane phospholipids by cytosolic phospholipase A2-alpha (cPLA2-alpha), which regulates cyclooxygenase- and lipoxygenase-mediated eicosanoid production by catalyzing the release of their substrate, AA. Sequencing of cPLA2-alpha cDNA demonstrated two heterozygous nonsynonymous single-base-pair mutations: Ser111Pro (S111P) and Arg485His (R485H), as well as a known single nucleotide polymorphism (SNP), Lys651Arg (K651R). CONCLUSIONS: Characterization of this cPLA2-alpha deficiency provides support for the importance of prostaglandins in protecting small intestinal integrity and indicates that loss of prostaglandin biosynthesis is sufficient to produce small intestinal ulcers.

Glucose homeostasis, insulin secretion, and islet phospholipids in mice that overexpress iPLA2beta in pancreatic beta-cells and in iPLA2beta-null mice.

Studies with genetically modified insulinoma cells suggest that group VIA phospholipase A(2) (iPLA(2)beta) participates in amplifying glucose-induced insulin secretion. INS-1 insulinoma cells that overexpress iPLA(2)beta, for example, exhibit amplified insulin-secretory responses to glucose and cAMP-elevating agents. To determine whether similar effects occur in whole animals, we prepared transgenic (TG) mice in which the rat insulin 1 promoter (RIP) drives iPLA(2)beta overexpression, and two characterized TG mouse lines exhibit similar phenotypes. Their pancreatic islet iPLA(2)beta expression is increased severalfold, as reflected by quantitative PCR of iPLA(2)beta mRNA, immunoblotting of iPLA(2)beta protein, and iPLA(2)beta enzymatic activity. Immunofluorescence microscopic studies of pancreatic sections confirm iPLA(2)beta overexpression in RIP-iPLA(2)beta-TG islet beta-cells without obviously perturbed islet morphology. Male RIP-iPLA(2)beta-TG mice exhibit lower blood glucose and higher plasma insulin concentrations than wild-type (WT) mice when fasting and develop lower blood glucose levels in glucose tolerance tests, but WT and TG blood glucose levels do not differ in insulin tolerance tests. Islets from male RIP-iPLA(2)beta-TG mice exhibit greater amplification of glucose-induced insulin secretion by a cAMP-elevating agent than WT islets. In contrast, islets from male iPLA(2)beta-null mice exhibit blunted insulin secretion, and those mice have impaired glucose tolerance. Arachidonate incorporation into and the phospholipid composition of RIP-iPLA(2)beta-TG islets are normal, but they exhibit reduced Kv2.1 delayed rectifier current and prolonged glucose-induced action potentials and elevations of cytosolic Ca(2+) concentration that suggest a molecular mechanism for the physiological role of iPLA(2)beta to amplify insulin secretion.