BACKGROUND AND PURPOSEw3-polyunsaturated fatty acids (w3-PUFAs) are known to exert anti-inflammatory effects in various disease models although their direct targets are only poorly characterized. EXPERIMENTAL APPROACHHere we report on two new cPLA2 inhibitors, the w3-derivatives AVX001 and AVX002, and their effects on inflammatory PGE2 production in cultures of renal mesangial cells. KEY RESULTSAVX001 and AVX002 dose-dependently inhibited the group IVA cytosolic phospholipase A2 (cPLA2) in an in vitro activity assay with similar IC50 values for AVX001 and AVX002, whereas the known cPLA2 inhibitor AACOCF3 was less potent and docosahexaenoic acid (DHA) was inactive. In renal mesangial cells, AVX001 and AVX002 suppressed IL-1b-induced PGE2 synthesis. Mechanistically, this effect occurred by a down-regulation of IL-1b-induced group IIA-sPLA2 protein expression, mRNA expression and promoter activity. A similar but less potent effect was seen with AACOCF3 and no effect was seen with DHA. As gene expression of sPLA2 is known to be regulated by the transcription factor NF-kB, we further investigated NF-kB activation. Both compounds prevented NF-kB activation by blocking degradation of the inhibitor of kB. CONCLUSIONS AND IMPLICATIONSThese data show for the first time that the novel cPLA2 inhibitors AVX001 and AVX002 exert an anti-inflammatory effect in cultures of renal mesangial cells and reduce the pro-inflammatory mediator PGE2 through an inhibitory effect on NF-kB activation. Therefore, these compounds may represent promising novel drugs for the treatment of inflammatory disorders. AbbreviationsAACOCF3, ATK, arachidonyl-trifluoromethyl ketone; AVX001, 1-octadeca-2,6,9,12,15-pentaenylsulfanyl-propan-2-one; AVX002, 1-octadeca-3,6,9,12,15-pentaenylsulfanyl-propan-2-one; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; IkB, inhibitor of kB; MAFP, methyl-arachidonyl fluorophosphonate; PAF-AH, PAF acetylhydrolase; PUFA, polyunsaturated fatty acid; RT-PCR, reverse transcriptase-PCR BJP British Journal of Pharmacology DOI:10.1111DOI:10. /j.1476DOI:10. -5381.2012 British Journal of Pharmacology (2012) IntroductionMesangial cells are specialized smooth muscle-like cells located in the renal glomerulus and are not only involved in the regulation of the glomerular filtration rate and in the preservation of the structural integrity of the glomerulus, but also play a central role in most pathological processes of the renal glomerulus (Kashgarian and Sterzel, 1992;Pfeilschifter, 1994;Gómez-Guerrero et al., 2005). Upon activation by a variety of pro-inflammatory cytokines, mesangial cells respond with three prominent reactions which are all hallmarks of many forms of glomerulonephritis: (i) increased proliferation; (ii) increased mediator production, including cytokines, chemokines, NO, superoxide radicals and PGs; and (iii) increased extracellular matrix production (Kashgarian and Sterzel, 1992;Pfeilschifter, 1994). The detailed mechanisms underlying these cellular responses are still not completely understood. One importan...

Both of the sphingosine kinase (SK) subtypes SK-1 and SK-2 catalyze the production of the bioactive lipid molecule sphingosine 1-phosphate (S1P). However, the subtype-specific cellular functions are largely unknown. In this study, we investigated the cellular function of SK-2 in primary mouse renal mesangial cells (mMC) and embryonic fibroblasts (MEF) from wild-type C57BL/6 or SK-2 knockout (SK2ko) mice. We found that SK2ko cells displayed a significantly higher proliferative and migratory activity when compared to wild-type cells, with concomitant increased cellular activities of the classical extracellular signal regulated kinase (ERK) and PI3K/Akt cascades, and of the small G protein RhoA. Furthermore, we detected an upregulation of SK-1 protein and S1P 3 receptor mRNA expression in SK-2ko cells. The MEK inhibitor U0126 and the S1P 1/3 receptor antagonist VPC23019 blocked the increased migration of SK-2ko cells. Additionally, S1P 3 ko mesangial cells showed a reduced proliferative behavior and reduced migration rate upon S1P stimulation, suggesting a crucial involvement of the S1P 3 receptor. In summary, our data demonstrate that SK-2 exerts suppressive effects on cell growth and migration in renal mesangial cells and fibroblasts, and that therapeutic targeting of SKs for treating proliferative diseases requires subtype-selective inhibitors.

Breast cancer is one of the most common and devastating malignancies among women worldwide. Recent evidence suggests that malignant progression is also driven by processes involving the sphingolipid molecule sphingosine 1-phosphate (S1P) and its binding to cognate receptor subtypes on the cell surface. To investigate the effect of this interaction on the metastatic phenotype, we used the breast cancer cell line MDA-MB-231 and the sublines 4175 and 1833 derived from lung and bone metastases in nude mice, respectively. In both metastatic cell lines expression of the S1P receptor was strongly upregulated compared to the parental cells and correlated with higher S1P-induced intracellular calcium ([Ca]), higher cyclooxygenase (COX)-2 and microsomal prostaglandin (PG) E synthase expression, and consequently with increased PGE synthesis. PGE synthesis was decreased by antagonists and siRNA against S1P and S1P. Moreover, in parental MDA-MB-231 cells overexpression of S1P by cDNA transfection also increased PGE synthesis, but only after treatment with the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine, indicating reversible silencing of the COX-2 promoter. Functionally, the metastatic sublines showed enhanced migration and Matrigel invasion in adapted Boyden chamber assays, which further increased by S1P stimulation. This response was abrogated by either S1P antagonism, COX-2 inhibition or PGE receptor 2 (EP) and 4 (EP) antagonism, but not by S1P antagonism. Our data demonstrate that in breast cancer cells overexpression of S1P and its activation by S1P has pro-inflammatory and pro-metastatic potential by inducing COX-2 expression and PGE signaling via EP and EP.

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