Cholesterol trafficking is required for mTOR activation in endothelial cells

Itraconazole Up-Regulates p27 Expression and Down-Regulates p21 Expression in Endothelial Cells.

Given that itraconazole causes cell cycle arrest in the G1 phase, we determined its effects on the expression of a number of known regulators of cell cycle progression at the G1-S transition. No appreciable changes were seen in the levels of CDK2, Cyclin D, and p53; however, the expression of Cyclin A, Cyclin E, and p21 was inhibited by itraconazole in a dose-dependent manner (Fig. 1). Strikingly, the level of p27 was up-regulated, rather than inhibited, by itraconazole (Fig. 1).

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Fig. 1.

Down-regulation of p21 and up-regulation of p27 by itraconazole in HUVECs. HUVECs were treated with itraconazole (ITRA) at the indicated concentrations for 24 h, and cell lysates were subjected to SDS/PAGE followed by Western blot analysis with the indicated antibodies. The relative band intensities of p27 normalized against those of tubulin from the corresponding lanes were provided.

Itraconazole Inhibits Both mTORC1 and mTORC2 in Endothelial Cells.

The opposing changes in the p27 and p21 levels, together with the G1 cell cycle arrest caused by itraconazole, were reminiscent of the effects of rapamycin (13), an inhibitor of mTOR that is also known to inhibit angiogenesis (14). We thus determined the effect of itraconazole on the mTOR pathway in endothelial cells. Like rapamycin, itraconazole inhibited the phosphorylation of p70S6K and 4E-BP1 in a dose-dependent manner (Fig. 2A ). In contrast, itraconazole had no effect on the phosphorylation of either ERK or JNK, demonstrating a high level of specificity for the mTOR pathway. In addition to p70S6K and 4E-BP1, which lie downstream of mTORC1, we examined the phosphorylation state of Akt, which lies downstream of mTORC2 (15). Unlike p70S6K, which suffered from a decrease in phosphorylation within half an hour of itraconazole treatment, phosphorylation of Akt at Ser473, which is activated by mTORC2 (15, 16), and Thr308 remained largely unchanged until 8 h after itraconazole treatment (Fig. 2B ). On prolonged exposure of endothelial cells to itraconazole, phosphorylation of Akt at both sites was inhibited by the drug in a dose-dependent manner (Fig. 2C ). These results suggested that itraconazole inhibited both mTORC1 and mTORC2, and inhibition of mTORC2 likely occurred as a secondary consequence of mTORC1 inhibition.

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Fig. 2.

Itraconazole (ITRA) inhibits both mTORC1 and mTORC2 in HUVECs. (A, C, and D) HUVECs were treated with ITRA and processed in the same manner as in Fig. 1. (B and E) HUVECs were treated with ITRA (2 μM) for the indicated time, and cell lysates were subjected to SDS/PAGE followed by Western blot analysis with the indicated antibodies.

In addition to blocking cell cycle progression in the G1 phase, rapamycin has been shown to induce autophagy in both yeast and mammalian cells (17, 18). We thus determined whether itraconazole was also capable of inducing autophagy in human umbilical vein endothelial cells (HUVECs). Microtubule-associated protein 1 light chain 3 (LC3) is a widely used marker of autophagy. During autophagy, the cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form a conjugate (LC3-II), which can then be recruited to the autophagosome membranes (19). When HUVECs were treated with itraconazole, we observed a dose-dependent and time-dependent induction of LC3-II (Fig. 2 D and E ). Using indirect immunofluorescence, we also found that LC3 exhibited a punctate staining pattern indicative of autophagosome formation on treatment of HUVECs with itraconazole (Fig. S1A ). Together, these results suggested that the antiangiogenic activity of itraconazole is attributable to its ability to inhibit the mTOR pathway. The different onset of inhibition of mTORC1 and mTORC2 by itraconazole is similar to the effects of rapamycin (20).

Itraconazole Inhibits Cholesterol Trafficking in Endothelial Cells.

The inhibitory effect of itraconazole on endothelial cell proliferation has been shown to be mediated, in part, through its inhibition of lanosterol 14α-demethylase, and hence de novo cholesterol biosynthesis (12). We thus wondered whether the inhibition of the mTOR pathway by itraconazole was also caused by cholesterol deprivation. Cyclodextrin was widely used to directly incorporate cholesterol to cell plasma membranes (21). When cholesterol/cyclodextrin complex was added to HUVECs, it partially reversed the inhibition of endothelial cell proliferation by itraconazole in normal cell growth medium (Fig. 3A ), suggesting that itraconazole caused a depletion of cholesterol on the plasma membrane of endothelial cells.

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Fig. 3.

Itraconazole causes accumulation of cholesterol in the late endosome/lysosome, and exogenous cholesterol partially rescues itraconazole inhibition of HUVEC proliferation and mTOR activity. (A) HUVECs were seeded into 96-well plates and incubated with the indicated drugs in the presence and absence of 20 μg/mL cholesterol, cyclodextrin (CD), or cholesterol/CD complex for 24 h. Cells were then pulsed with [³H]-thymidine for 6 h. Cell proliferation rates were normalized to those of cells treated with DMSO, cholesterol, CD, or cholesterol/CD complex only. (B) HUVECs were treated with 2 μM itraconazole for 24 h and then fixed for immunostaining. (C) HUVECs were treated with 2 μM itraconazole in the presence of CD or cholesterol/CD complex for 24 h, and cell lysates were subjected to SDS/PAGE followed by Western blot analyses. The values provided below the bottom of the gel are the relative intensities of the p-S6K1 normalized against those of the S6K1 bands.

To confirm the effect of itraconazole on the cholesterol level in plasma membrane further, we applied filipin, a specific fluorescent marker of unesterized cholesterol (22), to visualize and follow the distribution of free cholesterol in HUVECs in the absence and the presence of itraconazole. In control cells, cholesterol was clearly visible on the plasma membrane as well as on membranes of intracellular organelles such as the nuclear membrane and the ER (Fig. 3B ). As expected, there was a dramatic decrease in cholesterol on the plasma membrane under itraconazole treatment. Surprisingly, the majority of cholesterol appeared to be accumulated in an intracellular compartment in the cytosolic space (Fig. 3B ). The cholesterol-staining pattern was reminiscent of late endosomes and lysosomes. We thus performed double staining with LAMP-1, a marker of late endosome and lysosome (23), which revealed that free cholesterol was trapped within the late endosomal and lysosomal compartments by itraconazole. This was further confirmed by the lack of colocalization of free cholesterol and protein disulfide isomerase, an ER marker, or cytochrome C, a mitochondria marker (Fig. S1 B and C ). These observations suggested that itraconazole inhibited cholesterol trafficking out of the lysosomes and raised the possibility that inhibition of intracellular cholesterol trafficking can lead to inhibition of the mTOR pathway.

Proper Cholesterol Trafficking Is Essential for mTOR Activation in Endothelial Cells.

To assess the causal relation between inhibition of cholesterol trafficking by itraconazole and inhibition of the mTOR pathway, we treated HUVECs with itraconazole in the presence and absence of cyclodextran-cholesterol and determined the phosphorylation state of p70S6K. As shown in Fig. 3C , inhibition of phosphorylation of p70S6K by itraconazole was partially but significantly reversed by cyclodextran-cholesterol adduct but not by either cholesterol or cyclodextran alone. In addition, the induction of LC3-II and phosphorylation of 4E-BP1 by itraconazole were partially but significantly rescued by the cyclodextran-cholesterol complex (Fig. S2 A and B ). In contrast, the inhibition of mTOR by rapamycin in HUVECs was unaffected by the cyclodextran-cholesterol complex (Fig. S2C ). These results suggest that disruption of intracellular cholesterol trafficking from the endosome/lysosome to the plasma membrane is largely responsible for inhibition of the mTOR pathway.

Intracellular cholesterol trafficking has been shown to require two cholesterol binding proteins within the lumen of late endosome and lysosome, the Niemann–Pick disease type C (NPC)1 and NPC2 proteins (24 –26). As their names imply, genetic mutations in either gene have been shown to be associated with the corresponding neurological disease in humans and to cause blockade of intracellular cholesterol trafficking (27, 28). To determine whether inhibition of intracellular cholesterol trafficking by alternative means also caused inhibition of the mTOR pathway in endothelial cells, we knocked down NPC1 and NPC2, respectively, using siRNA in HUVECs (Fig. 4A ) and determined the effects on the phosphorylation of p70S6K. The siRNA oligonucleotides were identified that caused appreciable, albeit incomplete, down-regulation of NPC1 and NPC2 proteins in HUVECs by 24 h posttransfection. Similar to itraconazole, knockdown of NPC1 or NPC2 also led to the inhibition of p70S6K phosphorylation without significantly affecting the phosphorylation of several other kinases, including PKCα, ERK, and JNK (Fig. 4A and Fig. S2D ). In addition to dysregulating the functions of NPC1 or NPC2, two small molecule inhibitors of intracellular cholesterol trafficking are known, U18666A and imipramine (29). Both U18666A and imipramine have been shown to cause accumulation of cholesterol in the late endosome and lysosome. We thus determined their effects on endothelial cell proliferation and mTOR activity using phosphorylation of p70S6K as a readout. Both U18666A and imipramine inhibited HUVEC proliferation in a dose-dependent manner, with IC₅₀ values comparable to those for inhibition of cholesterol trafficking (Fig. S3 A and B ). They also exhibited a dose-dependent inhibition of p70S6K phosphorylation (Fig. 4 B and C ) with effective concentrations similar to those required for inhibiting intracellular cholesterol trafficking (Fig. S3C ). Similar to itraconazole, neither U18666A nor imipramine affected the phosphorylation of PKCα, ERK, or JNK, displaying high specificity for the mTOR signaling pathway (Fig. 4 B and C ).

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Fig. 4.

Blockade of cholesterol trafficking by siRNA knockdown of NPC1/2 or the small molecule inhibitors U18666A and imipramine leads to mTOR inhibition. (A) HUVECs were transfected with the indicated siRNA oligonucleotides for 24 h before they were lysed for Western blot analysis. (B and C) HUVECs were treated with the indicated drugs for 24 h before they were harvested for immunoblot analyses.

Among the known activators of the mTOR pathway are insulin and amino acids (30, 31). We thus determined the effects of itraconazole, U18666A, and imipramine on insulin- and amino acid-stimulated mTOR activation in HUVECs. All three inhibitors effectively inhibited the activation of mTOR induced by either insulin or amino acids (Fig. S4 A and B ). Thus, proper cholesterol trafficking is necessary for mTOR activation by multiple upstream signaling inputs in endothelial cells. In contrast to the treatment with small molecule inhibitors, however, knockdown of either NPC1 or NPC2 only had marginal effects on mTOR activation by insulin or amino acids (Fig. S4 C and D ). It is possible that the knockdown was incomplete and that the remaining NPC1 and NPC2 proteins were sufficient to support mTOR activation by insulin or amino acids.

The mTOR pathway has been shown to play a key role in protein synthesis by regulating cap-dependent translation (30, 32). We wondered whether cholesterol trafficking also regulates protein synthesis in endothelial cells. A luciferase reporter assay was thus performed. As shown in Fig. S5A , pateamine A (PatA), a known inhibitor of eukaryotic translation initiation, dramatically inhibited cap-dependent translation. Somewhat surprisingly, rapamycin had a much smaller effect on cap-dependent translation than PatA, which suggested that the dependence of cap-dependent translation on mTOR is not as pronounced in endothelial cells. The extent of inhibition of cap-dependent inhibition by rapamycin is in agreement with that observed previously (33). Nevertheless, itraconazole also had a small but significant effect on cap-dependent translation similar to rapamycin. Furthermore, knockdown of either NPC1 or NPC2 had a similar effect on cap-dependent translation (Fig. S5B ). The similar effects of itraconazole, rapamycin, and NPC1/2 knockdown on cap-dependent translation in endothelial cells are consistent with their common inhibition of mTOR.

Reversal of Itraconazole-Induced Accumulation of Cholesterol in Late Endosomes/Lysosomes and Inhibition of mTOR by Thapsigargin.

The NPC phenotype has recently been shown to be attributable to the dysregulation of endosomal/lysosomal calcium homeostasis, and thapsigargin, which releases ER calcium, was shown to correct the cellular NPC phenotype (34). We thus examined the effect of thapsigargin on cholesterol accumulation in endosomes/lysosomes caused by itraconazole. Thapsigargin restored the transport of cholesterol from endosomes/lysosomes to the plasma membrane in HUVECs (Fig. 5A ). We next determined whether thapsigargin treatment could reverse the inhibition of the mTOR pathway by itraconazole. Indeed, thapsigargin not only reversed inhibition of p70S6K phosphorylation by itraconazole but conferred resistance to both U18666A and imipramine (Fig. 5B ), indicating that restoration of cholesterol trafficking by thapsigargin confers resistance to mTOR inhibition by all three inhibitors of intracellular cholesterol transport. In contrast, inhibition of mTOR by rapamycin, which does not affect intracellular cholesterol trafficking, could not be reversed by thapsigargin (Fig. S6A ). In addition to its effect on ER calcium release, thapsigargin is known to induce unfolded protein response (35). We thus determined the ability of tunicamycin, another known inducer of unfolded protein response, to reverse the inhibition of the mTOR pathway by itraconazole, U18666A, and imipramine. Unlike thapsingargin, tunicamycin is incapable of reversing the inhibition of p70S6K phosphorylation by any of the three inhibitors (Fig. S6B ). Together, these results indicated that proper cholesterol trafficking is required for mTOR signaling in endothelial cells.

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Fig. 5.

Effects of thapsigargin (TG) and cholesterol/cyclodextrin complex on the inhibition of cholesterol trafficking and mTOR activity by itraconazole (ITRA), U18666A, and imipramine. (A and B) HUVECs were preincubated with 1 μM TG for 1 h before they were treated with 2 μM itraconazole, 30 μM imipramine, or 10 μM U18666A for an additional 24 h. They were then fixed for filipin staining (A) or harvested for immunoblot analysis (B). (C) HUVECs were incubated with 10 μM U18666A or 30 μM imipramine in the presence or absence of 4 μg/mL cholesterol/0.1% cyclodextrin complex for 24 h before being harvested for immunoblot analyses using the indicated antibodies.

In addition to thapsigargin, we determined the effect of exogenously administered cholesterol/cyclodextrin complex on the inhibition of mTOR by U18666A and imipramine. Similar to what was observed for itraconazole, the inhibition of mTOR by either U18666A or imipramine was significantly reduced in the presence of cholesterol/cyclodextrin, consistent with the notion that the inhibitory effects of those two compounds arose from their effects on cholesterol transport from the lysosome to the plasma membrane (Fig. 5C ).