Upregulation of the S1P3 receptor in metastatic breast cancer cells increases migration and invasion by induction of PGE2 and EP2/EP4 activation.

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 S1P3 receptor was strongly upregulated compared to the parental cells and correlated with higher S1P-induced intracellular calcium ([Ca2+]i), higher cyclooxygenase (COX)-2 and microsomal prostaglandin (PG) E2 synthase expression, and consequently with increased PGE2 synthesis. PGE2 synthesis was decreased by antagonists and siRNA against S1P3 and S1P2. Moreover, in parental MDA-MB-231 cells overexpression of S1P3 by cDNA transfection also increased PGE2 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 S1P3 antagonism, COX-2 inhibition or PGE2 receptor 2 (EP2) and 4 (EP4) antagonism, but not by S1P2 antagonism. Our data demonstrate that in breast cancer cells overexpression of S1P3 and its activation by S1P has pro-inflammatory and pro-metastatic potential by inducing COX-2 expression and PGE2 signaling via EP2 and EP4.

Sphingosine kinase 2 deficiency increases proliferation and migration of renal mouse mesangial cells and fibroblasts.

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 S1P3 receptor mRNA expression in SK-2ko cells. The MEK inhibitor U0126 and the S1P1/3 receptor antagonist VPC23019 blocked the increased migration of SK-2ko cells. Additionally, S1P3ko mesangial cells showed a reduced proliferative behavior and reduced migration rate upon S1P stimulation, suggesting a crucial involvement of the S1P3 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.

The ω3-polyunsaturated fatty acid derivatives AVX001 and AVX002 directly inhibit cytosolic phospholipase A(2) and suppress PGE(2) formation in mesangial cells.

BACKGROUND AND PURPOSE: ω3-polyunsaturated fatty acids (ω3-PUFAs) are known to exert anti-inflammatory effects in various disease models although their direct targets are only poorly characterized. EXPERIMENTAL APPROACH: Here we report on two new cPLA(2) inhibitors, the ω3-derivatives AVX001 and AVX002, and their effects on inflammatory PGE(2) production in cultures of renal mesangial cells. KEY RESULTS: AVX001 and AVX002 dose-dependently inhibited the group IVA cytosolic phospholipase A(2) (cPLA(2) ) in an in vitro activity assay with similar IC(50) values for AVX001 and AVX002, whereas the known cPLA(2) inhibitor AACOCF(3) was less potent and docosahexaenoic acid (DHA) was inactive. In renal mesangial cells, AVX001 and AVX002 suppressed IL-1ß-induced PGE(2) synthesis. Mechanistically, this effect occurred by a down-regulation of IL-1ß-induced group IIA-sPLA(2) protein expression, mRNA expression and promoter activity. A similar but less potent effect was seen with AACOCF(3) and no effect was seen with DHA. As gene expression of sPLA(2) is known to be regulated by the transcription factor NF-κB, we further investigated NF-κB activation. Both compounds prevented NF-κB activation by blocking degradation of the inhibitor of κB. CONCLUSIONS AND IMPLICATIONS: These data show for the first time that the novel cPLA(2) inhibitors AVX001 and AVX002 exert an anti-inflammatory effect in cultures of renal mesangial cells and reduce the pro-inflammatory mediator PGE(2) through an inhibitory effect on NF-κB activation. Therefore, these compounds may represent promising novel drugs for the treatment of inflammatory disorders.