Type II-secreted phospholipase A 2 (type II-sPLA 2 ) is expressed in smooth muscle cells during atherosclerosis or in response to interleukin-1␤. The present study shows that the induction of type II-sPLA 2 gene by interleukin-1␤ requires activation of the NFB pathway and cytosolic PLA 2 /PPAR␥ pathway, which are both necessary to achieve the transcriptional process. Interleukin-1␤ induced type II-sPLA 2 gene dose-and time-dependently and increased the binding of NFB to a specific site of type II-sPLA 2 promoter. This effect was abolished by proteinase inhibitors that block the proteasome machinery and NFB nuclear translocation. Type II-sPLA 2 induction was also obtained by free arachidonic acid and was blocked by either AACOCF 3 , a specific cytosolic-PLA 2 inhibitor, PD98059, a mitogen-activated protein kinase kinase inhibitor which prevents cytosolic PLA 2 activation, or nordihydroguaiaretic acid, a lipoxygenase inhibitor, but not by the cyclooxygenase inhibitor indomethacin, suggesting a role for a lipoxygenase product. Type II-sPLA 2 induction was obtained after treatment of the cells by 15-deoxy-⌬ 12,14 -dehydroprostaglandin J 2 , carbaprostacyclin, and 9-hydroxyoctadecadienoic acid, which are ligands of peroxisome proliferator-activated receptor (PPAR) ␥, whereas PPAR␣ ligands were ineffective. Interleukin-1␤ as well as PPAR␥-ligands stimulated the activity of a reporter gene containing PPAR␥-binding sites in its promoter. Binding of both NFB and PPAR␥ to their promoter is required to stimulate the transcriptional process since inhibitors of each class block interleukin-1␤-induced type II-sPLA 2 gene activation. We therefore suggest that NFB and PPAR␥ cooperate at the enhanceosome-coactivator level to turn on transcription of the proinflammatory type II-sPLA 2 gene.

The protein product of the growth arrest-specific gene 6 (Gas6) is a secreted ligand for tyrosine kinase receptors, among which Axl is the most widely distributed and displays the highest affinity for Gas6. The Gas6/Axl signaling pathway has been increasingly implicated in growth and survival processes occurring during development and tissue repair. In liver, after an acute or chronic injury, repair involves macrophages and hepatic stellate cells (HSC) activated into myofibroblastic cells (HSC/MFB), which produce cytokines and matrix proteins. We investigated the expression and the role of Gas6 and its receptor Axl in liver repair. Three days after CCl 4 -induced liver injury in the rat, we detected the expression of Gas6 in ED1-positive macrophages as well as in desmin-positive HSC, which accumulated in injured areas. Axl, the high-affinity receptor for Gas6, was detected in macrophages, HSC, and HSC/MFB. In vitro, expression of ␥-carboxylated Gas6 was strongly induced in HSC along with their transformation into myofibroblasts, and it exerted an anti-apoptotic effect on both HSC and HSC/MFB mediated by the Axl/PI3-kinase/Akt pathway. In conclusion, Gas6 is a survival factor for these cells and we suggest

ObjectiveSustained inflammation originating from macrophages is a driving force of fibrosis progression and resolution. Monoacylglycerol lipase (MAGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. It is a proinflammatory enzyme that metabolises 2-arachidonoylglycerol, an endocannabinoid receptor ligand, into arachidonic acid. Here, we investigated the impact of MAGL on inflammation and fibrosis during chronic liver injury.DesignC57BL/6J mice and mice with global invalidation of MAGL (MAGL -/- ), or myeloid-specific deletion of either MAGL (MAGLMye-/-), ATG5 (ATGMye-/-) or CB2 (CB2Mye-/-), were used. Fibrosis was induced by repeated carbon tetrachloride (CCl4) injections or bile duct ligation (BDL). Studies were performed on peritoneal or bone marrow-derived macrophages and Kupffer cells.ResultsMAGL -/- or MAGLMye-/- mice exposed to CCl4 or subjected to BDL were more resistant to inflammation and fibrosis than wild-type counterparts. Therapeutic intervention with MJN110, an MAGL inhibitor, reduced hepatic macrophage number and inflammatory gene expression and slowed down fibrosis progression. MAGL inhibitors also accelerated fibrosis regression and increased Ly-6Clow macrophage number. Antifibrogenic effects exclusively relied on MAGL inhibition in macrophages, since MJN110 treatment of MAGLMye-/- BDL mice did not further decrease liver fibrosis. Cultured macrophages exposed to MJN110 or from MAGLMye-/- mice displayed reduced cytokine secretion. These effects were independent of the cannabinoid receptor 2, as they were preserved in CB2Mye-/- mice. They relied on macrophage autophagy, since anti-inflammatory and antifibrogenic effects of MJN110 were lost in ATG5Mye-/- BDL mice, and were associated with increased autophagic flux and autophagosome biosynthesis in macrophages when MAGL was pharmacologically or genetically inhibited.ConclusionMAGL is an immunometabolic target in the liver. MAGL inhibitors may show promising antifibrogenic effects during chronic liver injury.

The Gas6/Axl pathway has been increasingly implicated in regeneration and tissue repair and, recently, in the control of innate immunity. In liver, we have demonstrated that Gas6 and its receptor Axl are expressed in macrophages, progenitor cells, and myofibroblasts and that Gas6 deficiency reduced inflammation and myofibroblast activation, causing delayed liver repair in response to acute injury. All these data suggest a role of Gas6/Axl signaling in pathogenesis of chronic liver diseases. In the present study, we address the role of Gas6 in steatohepatitis and progression to liver fibrosis using Gas6-deficient mice fed a choline-deficient ethionine-supplemented diet (CDE) or receiving a chronic carbon tetrachloride (CCl(4)) treatment. Gas6 deficiency attenuated hepatic steatosis by limiting CDE-induced downregulation of genes involved in β-oxidation observed in wild-type animals. Moreover, Gas6-deficient mice displayed reduction of hepatic inflammation, revealed by limited F4/80-positive macrophage infiltration, decreased expression of IL-1β, TNF-α, lymphotoxin-β, and monocyte chemotactic protein-1, and attenuated hepatic progenitor cell response to CDE diet. Gas6 deficiency reduced CDE-induced fibrogenesis and hepatic myofibroblast activation and decreased expression of TGF-β and collagen 1 mRNAs. After chronic CCl(4) injury, Gas6-deficient mice also exhibited reduced liver fibrosis as a consequence of defective macrophage recruitment compared with wild-type animals. We conclude that improvement of steatohepatitis and fibrosis in Gas6(-/-) mice is linked to an inhibition of the inflammatory response that controls lipid metabolism and myofibroblast activation. This study highlights the deleterious effect of Gas6 in the progression of steatosis to steatohepatitis and fibrosis.

The abundant secretion of type IIA secreted phospholipase A(2) (sPLA(2)) is a major feature of the inflammatory process of atherosclerosis. sPLA(2) is crucial for the development of inflammation, as it catalyses the production of lipid mediators and induces the proliferation of smooth muscle cells. We have analysed the activation of sPLA(2) transcription by cAMP and interleukin-1beta (IL-1beta), and shown that the 500 bp region upstream of the transcription start site of the rat sPLA(2) gene is implicated in activation by synergistically acting cAMP and IL-1beta. We transiently transfected and stimulated rat smooth muscle cells in primary culture and measured the promoter activities of serial and site-directed deletion mutants of sPLA(2)-luciferase constructs. A distal region, between -488 and -157 bp, bearing a CAAT/enhancer binding protein (C/EBP)-responsive element (-242 to -223) was sufficient for cAMP/protein kinase A-mediated sPLA(2) promoter activation. We find evidence for the first time that activation of the sPLA(2) promoter by IL-1beta requires activation of an Ets-responsive element in the -184 to -180 region of the distal promoter via the Ras pathway and a nuclear factor-kappaB site at positions -141 to -131 of the proximal promoter. We also used electrophoretic mobility shift assays to identify five binding sites for the Sp1 factor; a specific inhibitor of Sp1, mithramycin A, showed that this factor is crucial for the basal activity of the sPLA(2) promoter.

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There is good evidence that the n-3 polyunsaturated fatty acids (PUFAs) in fish oil have antiinflammatory effects and reduce the pathogenesis of atherosclerosis. However, the mechanisms underlying these actions are largely unknown. This study was designed to investigate the effects of membrane incorporation of two major components of fish oil [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)], on rat smooth muscle cells (SMCs) activation induced by interleukin-1 ␤ (IL1 ␤ ). We compared their effects with those of n-6 arachidonic acid (AA). Expression of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 adhesion molecules involved in SMCs migration was enhanced by AA, whereas EPA and DHA had no similar effects. We established that AA potentiates IL1 ␤ -induced expression of the type IIA secreted phospholipase A2 (sPLA2) gene, whereas EPA and DHA reduce this stimulation. EPA and DHA also abolished proinflammatory prostaglandin PGE2 production by inhibiting the IL1 ␤ -induced production of cyclooxygenase-2 (COX-2) mRNA. Much interest was then focused on three transcriptional factors implicated in inflammation control and especially in modulating rat sPLA2 and COX-2 gene transcription: nuclear factor-B, CCAAT/enhancer binding protein ␤ , and E26 transformation-specific-1. electrophoretic mobility shift assay revealed that the binding activity of all three factors was increased by AA and reduced (or not affected) by n-3 PUFA. These results indicate that EPA and DHA act in opposition to AA by modulating various steps of the inflammatory process induced by IL1 ␤ , probably by reducing mitogen-activated protein kinase p42/p44 activity. -Bousserouel, S., A. Brouillet, G. Béréziat, M. Raymondjean, and M. Andréani. Different effects of n-6 and n-3 polyunsaturated fatty acids on the activation of rat smooth muscle cells by interleukin-1 ␤ . J. Lipid Res. 2003. 44: 601-611.

Abstract-Type II secreted phospholipase A 2 (sPLA 2 ) releases precursors of important inflammatory lipid mediators from phospholipids. Some observations have indicated that the sPLA 2 , which has been implicated in chronic inflammatory conditions such as arthritis, contributes to atherosclerosis in the arterial wall. sPLA 2 was not detected in control vascular smooth muscle cells (VSMC). Treatment of VSMC with agents that increase intracellular cAMP (eg, forskolin, dibutyryl [db]-cAMP) resulted in a time-and concentration-dependent increase in sPLA 2 gene expression. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) showed a marked dose-dependent inhibition of forskolin-induced mRNA by protein kinase A inhibitor. Electrophoretic mobility shift analysis of nuclear proteins from forskolin-treated and db-cAMP-treated VSMC with C/EBP consensus oligonucleotides and C/EBP oligonucleotides from the rat promoter revealed greater binding than in control VSMC. Incubation of VSMC with H89, a specific protein kinase inhibitor, also blocked the binding of nuclear C/EBP to the C/EBP site of the rat promoter induced by db-cAMP and forskolin. Binding was unchanged with the use of CRE consensus oligonucleotides. Antibodies revealed the specific formation of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP-␤ and -␦ antibodies.Functional activation of C/EBP was confirmed by a luciferase reporter gene assay. A construct comprising 4 tandem repeat copies of the C/EBP element from the rat sPLA 2 promoter linked to luciferase was transcriptionally activated in VSMC by cotransfection with expression vector for the protein kinase A catalytic subunit. It was also significantly activated in transfected VSMC treated by forskolin or db-cAMP. H89 inhibited this activations. We therefore conclude that the increases in sPLA 2 mRNA and enzyme activity produced by cAMP-elevating agents is controlled by a mechanism involving nuclear C/EBP-␤ and -␦ acting through a protein kinase A signaling pathway. (Arterioscler