Figure 10. Effects of U18666A on the intracellular distribution of FC and LDL in HeLa cells and HMDM. HeLa cells and HMDM were respectively incubated for 6 h with 200 mg/mL LDL (A) or 50 mg/ ml AcLDL (C) with 3 mg/ml U18666A or without (control) and stained with filipin to detect FC. (B–D) HeLa cells and HMDM were respectively incubated for 6 h with 200 mg/ml DiI-LDL (B) or 50 mg/ml DiI-AcLDL (D) with 3 mg/ml U18666A or without (control) and processed to visualize LDL distribution. Images were obtained using wide-field epifluores- cence microscopy. doi:10.1371/journal.pone.0029042.g010 treated with dynasore, we observed a slight increase in the percentage of CE in U18666A-treated cells (Figures 11 C and D). The measure of CE in cells loaded with cholesterol in the presence the ACAT inhibitor revealed that the fraction of CE specifically generated by ACAT was 54% of the total intracellular pool of CE. When cells were treated with U18666A, the amount of CE generated by ACAT was decreased by 90%. Likewise the incorporation of myristate into CE was strongly inhibited by U18666A (Figure 11 E and F). Finally, we also measured the effects of U18666A on cholesterol efflux. As shown in Figures 12 A and D, the cellular cholesterol efflux to apoA-I, HDL, and MbCD was decreased by 42%, 58%, and 37%, respectively when macrophages were treated with U18666A. A similar decrease was measured in AcLDL-loaded macrophages after stimulation by the LXR/RXR agonists, 22OHC/9cRA (Figure 12B). Like dynasore, U18666A completely blocked the increase of ABCA1 and ABCG1 mRNA levels in response to the addition of AcLDL in HMDM (Figure 12C). Discussion Dynasore is a small drug that was identified through a chemical screen designed for inhibitors of the dynamin GTPase activity [10]. Because of its cell membrane permeability and reversibility, dynasore has since been extensively used to block in a selective and powerful manner the different endocytic pathways that rely on the dynamin GTPase. In a previous work based on the expression of Dynasore Impairs Cholesterol Homeostasis the K44A inactivated mutated form of dynamin, we could demonstrate that dynamin activity was also required for proper sorting of cholesterol in the endolysosomal network of HeLa cells. K44A dynamin expression led to the inhibition of free cholesterol endosomal egress to the ER, which instead accumulated into swollen endosomes of the endolysosomal network. As a result, the sterol-sensitive gene regulation was inhibited [9]. In this study, we took advantage of the cell permeability of dynasore to further extend our understanding of cholesterol intracellular trafficking in human macrophages. We chose to work on HMDM isolated from peripheral blood since available mouse or human macrophages cell lines do not entirely reproduce the characteristics of primary cultured HMDM. The monocytes were differentiated into macrophages by hM-CSF instead of hGM-CSF since it favors the expression of cell surface markers that are closer to those found in macrophages from atherosclerotic lesions [31]. AcLDL were used instead of native LDL since AcLDL uptake is 20-fold higher in human macrophages [32]. Indeed, the class A macrophage scavenger receptor SR-A accounts for the majority of AcLDL uptake, a pathway that is unregulated and can lead to the intracellular accumulation of large amounts of cholesterol and foam cell formation. In contrast, the uptake of native LDL is minor and regulated in macrophages [33]. Moreover, Wang et al. have shown in macrophages that AcLDL-delivered cholesterol is preferentially transported into the late endosomal network whereas LDL-derived cholesterol is preferentially transported to the recycling compartment [34]. In this study, we show that dynasore recapitulates the effects of the K44A dynamin mutant on cholesterol homeostasis that we have first described in HeLa cells. Dynasore, however, was more efficient to block LDL uptake than the K44A mutant, which probably reflects the more homogenous cell distribution of the drug. Interestingly, the inhibition of AcLDL uptake by dynasore was less efficient in human macrophages. This is in agreement with the lesser inhibition of AcLDL uptake that was also reported in HMDM after down-expression of clathrin [35]. Macrophages can use several alternative pathways such as macropinocytosis or caveolae for the uptake of AcLDL [35,36]. Our data suggest that the dynamin-independent endocytic pathways are the main contributors to AcLDL uptake in HMDM. In contrast to dynasore, U18666A leads to an increase of LDL uptake in both HeLa cells and HMDM. This increase, which is much higher in HMDM cells, can be inhibited by dynasore (not shown) indicating that U18666A up-regulates LDL uptake through the classical endocytic pathways. The increased LDL uptake is probably due to the known increased expression of LDL receptors resulting from the inhibition of FC delivery to the ER by U18666A. Dynasore treatment, which also leads to an increase of LDL receptor gene expression (Figure 4), does not result however in increased LDL uptake, because of its inhibitory effect on LDLR endocytosis through clathrin-coated pits. We show that dynasore blocks the delivery of exogenous LDL- derived cholesterol from the endolysosomal network to the ER, resulting in the inhibition of both sterol-sensitive genes regulation and cholesterol esterification. Similar findings were found in cells where dynamin 2 was down-expressed by RNAi treatment confirming the specificity of dynasore effect at the late endosomal network. Interestingly, dynasore and U18666A present the same effects on cholesterol membrane trafficking and sterol-sensitive genes regulation. The kinetics of the inhibition of LDL induced sterol-sensitive genes down-regulation showed that dynasore activity was maximal at 6 hours. We observed a progressive decrease of dynasore effect with time, an effect not observed with U18666A. Whether this is due to an inactivation of the drug with time or to the delivery of PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e29042