For confocal microscopy, BV 2 cells were plated onto 12 mm round cover slips and stained with an Alexa fluor CD11b antibody. We used 4,6 diamidino 2 phenylindole hydrochloride to identify nuclei in BV 2 cells. Statistical Gefitinib supplier analysis All data were expressed as the mean SD and analyzed by one way ANOVA followed by post hoc comparisons using the GraphPad Prism Version 4 software. P 0. 05 was considered statistically significant. Results sPLA2 IIA triggers microglial proliferation A great deal of attention has recently focused on the cytokine like actions of sPLA2 IIA and its input to inflammation associated diseases. Having been found highly expressed in several CNS pathological conditions, we hypothesized that sPLA2 IIA might act as a cytokine like modulator on brain resident immune cells.
To test this possibility, we examined whether Inhibitors,Modulators,Libraries sPLA2 IIA could induce some of the hallmarks of activated microglia. Inhibitors,Modulators,Libraries We used the immortalized mouse microglial cell line BV 2 as an in vitro model to mimic the microglial activation observed in neurodegenerative disorders �� such cells have been proven to reproduce the behavior of primary microglia Inhibitors,Modulators,Libraries and do not express endogenous sPLA2 IIA. Serum starved BV 2 cells were stimulated for 24 h with the indicated concentrations of sPLA2 Inhibitors,Modulators,Libraries IIA, and its effect on the proliferative activity of the cells was evaluated with a colorimetric assay. Our results revealed that sPLA2 IIA markedly stimulated cell proliferation in a dose dependent manner and reached a 3 fold increase when stimulated with 0. 5 ug ml of sPLA2 IIA, as compared with unstimulated cells.
The dose inducing the maximal change, 1 ug ml, was used for Inhibitors,Modulators,Libraries all subsequent experiments. We also found a strong mitogenic response to other secreted PLA2s, as well as to the well known inducer amplifier of microglia pro inflammatory functions, IFN��. Furthermore, as shown in Figure 1C, kinase inhibitor Nilotinib primary microglial cultures also responded to the addition of sPLA2 IIA and IFN�� with a modest but significant increase in cell proliferation. This effect on growth was paralleled by the activation phosphorylation of key proteins involved in cell survival and proliferation such as ERK, P70S6K and rS6. Acti vated forms of these proteins from whole cell lysates were monitored using specific anti phospho antibodies that recognize only their activated phosphorylated form. To determine whether the mTORC1 pathway was activated following sPLA2 IIA stimulation, we used an antibody that detects phosphorylation of P70S6K on threonine 389, a site well known to be selectively phos phorylated by mTORC1 and widely used to monitor mTORC1 activation. As shown in Figure 1D, sPLA2 IIA treatment induced a rapid and sustained increase in ERK, P70S6K and rS6 phosphorylation in BV 2 cells.