Dynasore Impairs Cholesterol Homeostasis Figure 12. U18666A impairs cellular cholesterol efflux from HMDM. Cells were incubated with 100 mg/ml AcLDL for 6 h and treated with 3 mg/ml U18666A or without (control). The cellular cholesterol efflux to 10 mg/ml apoA-I or 15 mg/ml HDL-PL before (A) and after (B) stimulation of ABCA1 and ABCG1 expression by the LXR/RXR agonists was quantified. Results are expressed as the percentage of the quantity of released cellular cholesterol into the medium to the total quantity of cholesterol in cells and medium. Each value is the mean of triplicate experiments. (C) Relative quantification of ABCA1 and ABCG1 transporter genes levels expressed as fold-variation over control (DMSO/LPDS) after normalization. All CT determinations were made in triplicate. (D) Passive cholesterol efflux to 1 mg/ml MaˆCD was quantified as above. doi:10.1371/journal.pone.0029042.g012 net cholesterol efflux from macrophages to HDL or serum [34,37,38]. Our data show that the cholesterol efflux mediated by ABCA1 and ABCG1 was impaired in dynasore-treated HMDM, even after stimulation with the LXR/RXR agonist. Similar results were obtained with U18666A. It has been shown that the endolysomal sequestration of LDL-derived cholesterol in NPC2/2 cells altered cholesterol efflux by reducing the amount of cholesterol substrate available for ABCA1 and by defective synthesis of LDL-cholesterol-derived side-chain oxysterols [39,40]. The decreased production of oxysterols, which are endogenous LXR ligands, results in reduced ABCA1 and ABCG1 expression and lower cholesterol efflux activity [41]. 27 hydro- xycholesterol is the most abundant oxysterol present in macro- phages. A key step in 27 hydroxycholesterol synthesis is the delivery of cholesterol to mitochondria where is present the sterol- 27 hydroxylase CYP27. It is therefore tempting to speculate that the decrease of ABCA1 and ABCG1 gene expression is due to a decreased delivery of cholesterol to mitochondria. Alternatively, the decrease of cholesterol efflux mediated by ABCA1 and ABCG1 could be the consequence of a reduction of the pool of FC at the plasma membrane since it is a preferential site to collect FC coming from the endolysosomal compartment. The role of dynamin in endolysosomal sorting remains poorly documented. We show here that dynasore treatment affects not only cholesterol trafficking but also the intracellular distribution of the M6PR. A recent work has shown that dynamin 2 controlled the exit of the EGFR from late endosomes through its association with CIN85 [42]. Altogether these data demonstrate that dynamin controls trafficking events within the endolysosomal system presumably through the scission of vesicular buds originating from early and/or late endosomes and affects several cargos that use this pathway. In conclusion, we have shown that the pharmacological inhibition of the dynamin GTPase activity by dynasore leads rapidly to the abnormal endosomal sequestration of FC and LDL, resulting in defective sterol-sensitive genes regulation and cholesterol efflux in HMDM. These results demonstrate that dynasore can be used to block the egress of FC from the endolysosomal network. Dynasore therefore represents an inter- esting alternative to U18666A and will be useful to better understand the complexity of cholesterol trafficking and homeo- stasis at the late endosomal interface. Author Contributions Conceived and designed the experiments: EG JLP NF CL BV. Performed the experiments: EG BV. Analyzed the data: EG CL BV. Contributed reagents/materials/analysis tools: PB LJ CL BV. Wrote the paper: EG CL BV. PLoS ONE | www.plosone.org 12 December 2011 | Volume 6 | Issue 12 | e29042