br Materials and methods br Results br

Materials and methods
Results
Discussion Macrophages and dendritic cells respond to Toll-like receptor (TLR) ligands by upregulating CH25H expression [4], [5]. In the latter cell type TLR-dependent upregulation is mediated via a signalling pathway that involves NFκB and IFNβ secretion and converges on activation of the JAK/STAT1 pathway [4]. In vivo, exposure of mice to the selective TLR4 agonist KDO induced strong upregulation of CH25H in SR 3576 [26]. Results of the present study revealed upregulated CH25H transcription and translation in GBM cells. Interestingly, within the NCI60 data set contained in the BioGPS gene portal GBM cells appear to take a unique role in terms of highest CH25H expression (http://biogps.org). The response to TNFα and IL1β was different with respect to cytokine selectivity in the two cell lines studied here (Fig. 1). Whether this is due to different cytokine receptor expression levels is currently not clear. However, both cytokines used here may activate JAK/STAT pathways. TNFα activates JAK/STAT1 [42] and IL1β activates STAT1 [43] and a STAT-like factor, leading to activation of gene transcription [44]. Therefore our observations are compatible with a pathway inducing transcriptional activation of CH25H identified in dendritic cells and macrophages [4]. In line with findings reported for lipopolysaccharide activated macrophages [5] we have observed efficient secretion of 25-OHC into the cellular supernatant. In experiments analyzing free transfer of cholesterol or 25-OHC from erythrocytes and plasma, exchange of 25-OHC was found to occur about 2000 times faster than that of cholesterol [45]. The rate of transfer of oxysterols from a monolayer to acceptor particles followed a clear rank order with the highest rate of transfer observed for 25-OHC [46]. As reported for the quantitatively dominating oxysterols 24S- and 27-OHC, also 25-OHC is transported in association with circulating lipoproteins including high-density lipoproteins [47]. This might be of functional importance for gliomagenesis since high-density lipoproteins containing sphingosine-1-phosphate (a potent lipid GBM mitogen; [48]) induced increased DNA synthesis, ERK phosphorylation and Ca2+ mobilization in glioma cells [49]. Significant progress has been made in elucidating the functional significance of oxysterol-induced B- and T-cell migration in lymphoid organs [29], [30], [50], [51], [52]. It is now widely accepted that EBI2-mediated chemotaxis represents an important molecular mechanism directing follicular B cell migration and localization. Of note, oxysterols represent natural ligands for EBI2 activation [29], [30], [51]. In line with findings from the present study (Fig. 6), PTX suppressed 7α,25-OHC stimulated and EBI2-mediated binding of GTP [30]. However, PTX-insensitive ERK activation in response to EBI2 engagement was also reported [52]. The functional potency of different oxysterols towards EBI2 is 7α,25-OHC>7α,27-OHC>7α–OHC>25-OHC>27-OHC [50]. Of relevance for the present study 25-OHC also confers agonist activity towards EBI2 albeit with lower affinity [32], [33]. In line, silencing of EBI2 using 21-mer siRNA reduced 25-OHC-induced THP-1 migration by 46% (Fig. 7). Whether EBI2 is responsible for increased migration of primary human monocytes in response to 25-OHC remains to be elucidated. The reason why mock transfection (GeneMute) and transfection with scrambled siRNA reduced migration of THP-1 cells in response to 25-OHC is currently not clear. However, lipid-based transfection reagents impact on cellular phospholipid composition (enrichment in 16:0 or 22:6-containing phosphatidylcholine species; unpublished observation; S.F.), alterations that could account for different receptor responsiveness. To get an indication whether GBM-derived 25-OHC could act as a chemotactic signal for monocytes, lipid extracts of GM133 media were used in THP-1 migration assays. Although an indirect approach, these experiments revealed that medium lipid extracts induced monocyte migration in a quantitatively comparable manner as identical concentrations of exogenously added 25-OHC (Fig. 6). A question remaining is how 25-OHC contributes to monocyte attraction in the tumor environment. Association of secreted 25-OHC with lipoproteins would be a plausible explanation. In light of the facts that HDL particles transport 25-OHC in the circulation [47], shuttle oxysterols between the circulation and the brain [2] and are potent effectors of the monocytic migratory response [53] this hypothesis could be reasonable. Silva and colleagues [54] showed that an oxysterol-containing lipid fraction isolated from osteoblast-conditioned medium potently induces migration of human breast cancer cells, raising the possibility that oxysterols even contribute to metastasis.