PPARc Ligands Differentially Impact OVCAR Cells Figure 9. Effect of PPARc antagonists on Ovcar3 proliferation. Cells were treated with GW9662 (A) or T007 (B) for 24 h and cell proliferation was assessed. Results are the means 6 SEM for at least 4 measurements. Bars that do not share a letter designation are significantly different (p,0.05). doi:10.1371/journal.pone.0016179.g009 decrease in the steady state levels of the protein in endothelial cells [24,25]. This is hypothesized to occur because TZDs mediate changes in the phosphorylation state of PPARc by MEK which leaves PPARc susceptible to degradation [47]. In light of this information, our protein data could be explained by the fact that TZDs cause differential degradation of PPAR(after activation and that the turn-over rate of PPAR(protein in cells treated with TGZ is faster than that of CGZ and Rosi resulting in a decrease in protein levels. In addition, it is possible that Rosi increases the protein stability of PPAR(whilst the effects on proliferation after treatment with CGZ and TGZ are PPAR(independent. Alterna- tively, TZDs may cause changes in the protein itself, such as phosphorylation, which alters other actions of the protein rather than just changing its overall transactivation as recently been shown by Choi et al. [48]. In light of our results that showed a lack of correlation between the levels of PPAR(mRNA and protein after TZD treatment, we explored the changes in activation of a PPAR(reporter as an index of PPAR(activity after TZD exposure. Surprisingly, PPAR(activity was not stimulated by any of the 4 TZDs after 24 h of treatment, in contrast to previous reports in other ovarian cancer cell lines that 50 mM of CGZ increased luciferase activity of a PPRE reporter by 18 h [21]. Based on this previous report, we speculated that promoter activation might be occurring at earlier or later time Figure 10. Effect of CGZ and TGZ treatment on Ovcar3 cell proliferation in cells transfected with DN or OE PPARc constructs. Ovcar3 cells were double transfected, serum starved for 24 hours and treated for an additional 24 hours with vehicle control (DMSO) or10 mM of CGZ or TGZ. Cell proliferation was assessed with a BrdU assay. Results are the means 6 SEM for at least 9 measurements from three individual experiments. Bars that do not share a letter designation are significantly different within a treatment group (p,0.05). doi:10.1371/journal.pone.0016179.g010 points. Hence, we tested the effect of TZDs on promoter activity at 4, 8, 24 and 32 hours. There were modest increases in luciferase activity in cells treated with CGZ at 8 and 32 hours (data not shown), which were insufficient to account for the changes in PPAR(expression. Discordance between protein levels and PPAR(activity has been previously seen in breast cancer cells but the exact mechanism underlying this difference remains unknown [34]. However, such an observation suggests that analysis of only PPARc protein levels may mistakenly be used to attribute cellular effects or responsiveness to treatments. The TZDs did not increase endogenous PPAR activity in these cells, however, TZD treatment increased luciferase activity when PPARc was overexpressed in Ovcar3 cells. This indicates that these cells constitutively activate PPARc and, when the receptor is highly abundant, further activation is possible. This activation is not limited by RXR, since 9-cis-RA does not affect the PPRE activity in Ovcar3 cells when transfected with empty, DN, or wildtype PPARc vectors. The difference in endogenous ligands or activating capacity between cells could lead to an increase in endogenous transactivation and potentially, an increased receptor degradation rate. Therefore, cellular levels of PPARc protein do not reflect the total activity of PPARc or the capacity of exogenous ligands to overcome endogenous PPARc activation. The present study explored whether proliferation and cell cycle arrest observed after TZD treatment were due directly to PPARc. Previously, the PPARc antagonists T007 and GW9662 were used to determine whether the effects of TZDs are PPARc dependent [20,49]. Our results showed no changed in cell proliferation when cells were treated with these two antagonists alone. When cells were treated with the antagonists in combination with TZDs, there were differential changes in cell proliferation. However, there are controversies regarding the specificity of PPARc antagonists [9,26], in particular that they can activate, rather than inactivate the PPRE reporter in normal breast cell lines [9]. Antagonists may also have PPARc independent effects [26]. Due to these potential confounding non-PPARc actions of the PPARc antagonists, we used an alternative molecular approach to determine whether TZDs act through PPARc. To accomplish this, we transfected Ovcar3 cells with a dominant negative form of PPARc or a wild type overexpression PPARc construct. The DN form of PPARc provided a more robust decrease in PPRE when compared to shRNA (data not shown). The observation that the DN decreased PPRE promoter activity again illustrates that there is endogenous transactivation of PPARc taking place in these cells. In addition, the finding that the TZDs cause an increase in reporter activity when the level of PPARc is overexpressed demonstrates that TZDs convey part of their activity through PPARc when there are sufficient, unoccupied receptors available. One can argue that the effect on proliferation may be due to the TZDs competing for the receptor with the endogenous ligand and recruiting different co- activators [42]; however, all four TZDs showed a similar trend in terms of luciferase activity indicating the possibility that they may convey some actions through PPARc but the effects on proliferation may not be PPARc dependent. This was evident when Ovcar3 cells were concurrently transfected with the wild type or DN PPARc, treated with CGZ or TGZ, and cell proliferation still decreased, indicating that the effects of CGZ and TGZ are not PPARc dependent. However the possibility exists that, even in the presence of the DN, there is still sufficient PPARc PLoS ONE | www.plosone.org 11 January 2011 | Volume 6 | Issue 1 | e16179