Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype.

BACKGROUND: Extracellular lysophosphatidic acid (LPA) species transmit signals via six different G protein-coupled receptors (LPAR1-6) and are indispensible for brain development and function of the nervous system. However, under neuroinflammatory conditions or brain damage, LPA levels increase, thereby inducing signaling cascades that counteract brain function. We describe a critical role for 1-oleyl-2-hydroxy-sn-glycero-3-phosphate (termed "LPA" throughout our study) in mediating a motile and pro-inflammatory microglial phenotype via LPAR5 that couples to protein kinase D (PKD)-mediated pathways. METHODS: Using the xCELLigence system and time-lapse microscopy, we investigated the migrational response of microglial cells. Different M1 and M2 markers were analyzed by confocal microscopy, flow cytometry, and immunoblotting. Using qPCR and ELISA, we studied the expression of migratory genes and quantitated the secretion of pro-inflammatory cytokines and chemokines, respectively. Different transcription factors that promote the regulation of pro-inflammatory genes were analyzed by western blot. Reactive oxygen species (ROS) and nitric oxide (NO) production, phagocytosis, and microglial cytotoxicity were determined using commercially available assay kits. RESULTS: LPA induces MAPK family and AKT activation and pro-inflammatory transcription factors' phosphorylation (NF-κB, c-Jun, STAT1, and STAT3) that were inhibited by both LPAR5 and PKD family antagonists. LPA increases migratory capacity, induces secretion of pro-inflammatory cytokines and chemokines and expression of M1 markers, enhances production of ROS and NO by microglia, and augments cytotoxicity of microglial cell-conditioned medium towards neurons. The PKD family inhibitor blunted all of these effects. We propose that interference with this signaling axis could aid in the development of new therapeutic approaches to control neuroinflammation under conditions of overshooting LPA production. CONCLUSIONS: In the present study, we show that inflammatory LPA levels increased the migratory response of microglia and promoted a pro-inflammatory phenotype via the LPAR5/PKD axis. Interference with this signaling axis reduced microglial migration, blunted microglial cytotoxicity, and abrogated the expression and secretion of pro-inflammatory mediators.

Inhibition of lung carcinoma cell growth by high density lipoprotein-associated alpha-tocopheryl-succinate.

Alpha-tocopheryl-succinate (alphaTS) is a synthetic, anti-neoplastic derivative of alpha-tocopherol. Here we studied the effects of free and high-density lipoprotein subclass 3 (HDL3)-associated alphaTS on the growth of human (A549) and mouse Lewis (LL2) lung carcinoma cells. Both free and HDL3-associated alphaTS inhibited A549 growth in a time- and concentration-dependent manner. Treatment of A549 cells with alphaTS-enriched HDL3 led to DNA fragmentation and a time-dependent decrease in immunoreactivity of poly(ADP-ribose)polymerase. Uptake experiments revealed a high capacity for selective alphaTS uptake in excess of holoparticle endocytosis. Overexpression of scavenger receptor class B, type I (SR-BI), the prime receptor mediating selective lipid uptake, in A549 cells resulted in significantly increased selective alphaTS uptake, a finding associated with complete cellular growth arrest. The present in vitro findings were verified in an in vivo model: tumor inoculation in C57BL6 was performed with either wild-type, beta-galactosidase- or SR-BI-overexpressing LL2 cells. After tumor inoculation, the animals received six consecutive intravenous injections of alphaTS. This experimental setup resulted in significantly reduced tumor burden in animals that were inoculated with SR-BI-overexpressing LL2 cells but not in animals inoculated with wild-type or beta-galactocidase-transfected cells. Based on our in vitro and in vivo findings, we propose that SR-BI could provide a novel route for HDL3-mediated drug delivery of anti-neoplastic drugs.

Modulation of microglial superoxide production by alpha-tocopherol in vitro: attenuation of p67(phox) translocation by a protein phosphatase-dependent pathway.

As in other phagocytic cells, the NADPH-oxidase system in microglia is thought to be primarily responsible for the production of superoxide anion radicals (O2(-.), a potentially cytotoxic reactive oxygen species. The assembly of a functional NADPH-oxidase complex at the plasma membrane depends on the phosphorylation and subsequent translocation of several cytosolic subunits. Immunocytochemical and subcellular fractionation experiments performed during the present study revealed that the NADPH-oxidase subunit p67(phox) translocates from the cytosol to the plasma membrane upon stimulation. Pre-incubation of microglia in alpha-tocopherol (alphaTocH) containing medium decreased O2(-.) production in a time- and concentration-dependent manner, findings attributed to attenuated p67(phox) translocation to the plasma membrane. Moreover, alphaTocH-supplementation of the culture medium resulted in decreased microglial protein kinase C (PKC) activities, an effect that could be partially or completely reversed by the addition of protein phosphatase inhibitors (okadaic acid and calyculin A). The addition of the PKC-inhibitor staurosporine inhibited the microglial respiratory burst in a manner comparable to alphaTocH. The addition of okadaic acid or calyculin A completely restored O2(-.) production in alphaTocH-supplemented cells. The present findings suggest that alphaTocH inactivates PKC via a PP1 or PP2A-mediated pathway and, as a consequence, blocks the phosphorylation-dependent translocation of p67(phox) to the plasma membrane. As a result, O2(-.) production by the microglial NADPH-oxidase system is substantially inhibited.

Lipoprotein lipase mediates the uptake of glycated LDL in fibroblasts, endothelial cells, and macrophages.

The nonenzymatic glycation of LDL is a naturally occurring chemical modification of apolipoprotein (apo)-B lysine residues by glucose. Once glycated, LDL is only poorly recognized by lipoprotein receptors including the LDL receptor (LDL-R), the LDL-R-related protein (LRP), and scavenger receptors. Glycated LDL (gLDL) is a preferred target for oxidative modifications. Additionally, its presence initiates different processes that can be considered "proatherogenic." Thus, LDL glycation might contribute to the increased atherosclerotic risk of patients with diabetes and familial hypercholesterolemia. Here we investigate whether lipoprotein lipase (LPL) can mediate the cellular uptake of gLDL. The addition of exogenous LPL to the culture medium of human skin fibroblasts, porcine aortic endothelial cells, and mouse peritoneal macrophages enhanced the binding, uptake, and degradation of gLDL markedly, and the relative effect of LPL on lipoprotein uptake increased with the degree of apoB glycation. The efficient uptake of gLDL by LDL-R-deficient fibroblasts and LRP-deficient Chinese hamster ovary cells in the presence of LPL suggested a mechanism that was independent of the LDL-R and LRP. In macrophages, the uptake of gLDL was also correlated with their ability to produce LPL endogenously. Mouse peritoneal macrophages from genetically modified mice, which lacked LPL, exhibited a 75% reduction of gLDL uptake compared with normal macrophages. The LPL-mediated effect required the association of the enzyme with cell surface glycosaminoglycans but was independent of its enzymatic activity. The uptake of gLDL in different cell types by an LPL-mediated process might have important implications for the cellular response after gLDL exposure as well as the removal of gLDL from the circulation.

The human breast carcinoma cell line HBL-100 acquires exogenous cholesterol from high-density lipoprotein via CLA-1 (CD-36 and LIMPII analogous 1)-mediated selective cholesteryl ester uptake.

Aberrant cell proliferation is one of the hallmarks of carcinogenesis, and cholesterol is thought to play an important role during cell proliferation and cancer progression. In the present study we examined the pathways that could contribute to enhanced proliferation rates of HBL-100 cells in the presence of apolipoprotein E-depleted high-density lipoprotein subclass 3 (HDL(3)). When HBL-100 cells were cultivated in the presence of HDL(3) (up to 200 microg/ml HDL(3) protein), the growth rates and cellular cholesterol content were directly related to the concentrations of HDL(3) in the culture medium. In principle, two pathways can contribute to cholesterol/cholesteryl ester (CE) uptake from HDL(3), (i) holoparticle- and (ii) scavenger-receptor BI (SR-BI)-mediated selective uptake of HDL(3)-associated CEs. Northern- and Western-blot analyses revealed the expression of CLA-1 (CD-36 and LIMPII analogous 1), the human homologue of the rodent HDL receptor SR-BI. In line with CLA-1 expression, selective uptake of HDL(3)-CEs exceeded HDL(3)-holoparticle uptake between 12- and 58-fold. Competition experiments demonstrated that CLA-1 ligands (oxidized HDL, oxidized and acetylated low-density lipoprotein and phosphatidylserine) inhibited selective HDL(3)-CE uptake. In line with the ligand-binding specificity of CLA-1, phosphatidylcholine did not compete for selective HDL(3)-CE uptake. Selective uptake was regulated by the availability of exogenous cholesterol and PMA, but not by adrenocorticotropic hormone. HPLC analysis revealed that a substantial part of HDL(3)-CE, which was taken up selectively, was subjected to intracellular hydrolysis. A potential candidate facilitating extralysosomal hydrolysis of HDL(3)-CE is hormone-sensitive lipase, an enzyme which was identified in HBL-100 cells by Western blots. Our findings demonstrate that HBL-100 cells are able to acquire HDL-CEs via selective uptake. Subsequent partial hydrolysis by hormone-sensitive lipase could provide 'free' cholesterol that is available for the synthesis of cellular membranes during proliferation of cancer cells.

Lipoprotein-associated alpha-tocopheryl-succinate inhibits cell growth and induces apoptosis in human MCF-7 and HBL-100 breast cancer cells.

alpha-Tocopheryl succinate (alpha-TS) is a potent inhibitor of tumor cell proliferation. The goal of the present study was to investigate whether and to what extent alpha-TS associates with plasma lipoproteins and if alpha-TS-enriched lipoproteins inhibit breast cancer cell growth in a manner comparable to the free drug. In vitro enrichment of human plasma revealed that alpha-TS readily associated with the main lipoprotein classes, findings confirmed in vivo in mice. At the highest alpha-TS concentrations, lipoproteins carrying 50000 (VLDL), 5000 (LDL) and 700 (HDL) alpha-TS molecules per lipoprotein particle were generated. alpha-TS enrichment generated lipoprotein particles with slightly decreased density and increased particle radius. To study whether the level of LDL-receptor (LDL-R) expression affects alpha-TS uptake from apoB/E containing lipoprotein particles human breast cancer cells with low (MCF-7) and normal (HBL-100) LDL-R expression were used. The uptake of free, VLDL- and (apoE-free) HDL(3)-associated alpha-TS was nearly identical for both cell lines. In contrast, uptake of LDL-associated alpha-TS by HBL-100 cells (normal LDL-R expression) was about twice as high as compared to MCF-7 cells (low LDL-R expression). VLDL and LDL-associated alpha-TS inhibited proliferation most effectively at the highest concentration of alpha-TS used (100% inhibition of MCF-7 growth with 20 microg/ml of lipoprotein-associated alpha-TS). However, also alpha-TS-free VLDL and LDL inhibited HBL-100 cell proliferation up to 55%. In both cell lines, alpha-TS-enriched HDL(3) inhibited cell growth by 40-60%. Incubation of both cell lines in the presence of free or lipoprotein-associated alpha-TS resulted in DNA fragmentation indicative of apoptosis. Collectively, the present findings demonstrate that: (1) alpha-TS readily associates with lipoproteins in vitro and in vivo; (2) the lipoprotein-enrichment efficacy was dependent on the particle size and/or the triglyceride content of the lipoprotein; (3) uptake of LDL-associated alpha-TS was apparently dependent on the level of LDL-R expression; and (4) lipoproteins were efficient alpha-TS carriers inducing reduced cell proliferation rates and apoptosis in human breast cancer cells as observed for the free drug.