Cholesterol trafficking is required for mTOR activation in endothelial cells

Department of Pharmacology and Oncology, The Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/2010; 107(10):4764-9. DOI: 10.1073/pnas.0910872107
Source: PubMed


Mammalian target of rapamycin (mTOR) constitutes a nodal point of a signaling network that regulates cell growth and proliferation in response to various environmental cues ranging from growth factor stimulation to nutrients to stress. Whether mTOR is also affected by cholesterol homeostasis, however, has remained unknown. We report that blockade of cholesterol trafficking through lysosome by a newly identified inhibitor of angiogenesis, itraconazole, leads to inhibition of mTOR activity in endothelial cells. Inhibition of mTOR by itraconazole but not rapamycin can be partially restored by extracellular cholesterol delivered by cyclodextrin. Moreover, other known inhibitors of endosomal/lysosomal cholesterol trafficking as well as siRNA knockdown of Niemann-Pick disease type C (NPC) 1 and NPC2 also cause inhibition of mTOR in endothelial cells. In addition, both the accumulation of cholesterol in the lysosome and inhibition of mTOR caused by itraconazole can be reversed by thapsigarin. These observations suggest that mTOR is likely to be involved in sensing membrane sterol concentrations in endothelial cells, and the cholesterol trafficking pathway is a promising target for the discovery of inhibitors of angiogenesis.

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    Archives of Pharmacal Research 07/2015; 38(9). DOI:10.1007/s12272-015-0628-1 · 2.05 Impact Factor
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    • "Conversely, Fang et al. demonstrated that AIBP mediates cholesterol efflux from endothelial cells, and its overexpression suppresses angiogenesis in animal models through inhibition of VEGFR2 signaling [19]. In our previous studies, itraconazole was found to inhibit cholesterol trafficking in endothelial cells [17]. Itraconazole is undergoing multiple clinical studies as an anti-angiogenic agent. "
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    ABSTRACT: Selective estrogen receptor modulators (SERM) including tamoxifen are known to inhibit angiogenesis. However, the underlying mechanism, which is independent of their action on the estrogen receptor (ER), has remained largely unknown. In the present study, we found that tamoxifen and other SERM inhibited cholesterol trafficking in endothelial cells, causing a hyper-accumulation of cholesterol in late endosomes/lysosomes. Inhibition of cholesterol trafficking by tamoxifen was accompanied by abnormal subcellular distribution of vascular endothelial growth factor receptor-2 (VEGFR2) and inhibition of the terminal glycosylation of the receptor. Tamoxifen also caused perinuclear positioning of lysosomes, which in turn trapped the mammalian target of rapamycin (mTOR) in perinuclear region in endothelial cells. Abnormal distribution of VEGFR2 and mTOR and inhibition of VEGFR2 and mTOR activities by tamoxifen were significantly reversed by addition of cholesterol-cyclodextrin complex to the culture media of endothelial cells. Moreover, high concentrations of tamoxifen inhibited endothelial and breast cancer cell proliferation in a cholesterol-dependent, but ER-independent, manner. Together, these results unraveled a previously unrecognized mechanism of angiogenesis inhibition by tamoxifen and other SERM, implicating cholesterol trafficking as an attractive therapeutic target for cancer treatment. Copyright © 2015. Published by Elsevier Ireland Ltd.
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    • "The antiangiogenic activity of ITZ has been attributed at least in part to its inhibition of the mTOR signaling pathway through disruption of the shuttling of cholesterol between plasma membrane and late endosomes/lysosomes, thereby inducing the accumulation of cholesterol in the endolysosomal system (Xu et al., 2010). We found that cholesterol, stained with filipin, was redistributed not only by ITZ and posaconazole but also by ketoconazole (which does not inhibit virus replication) in two human cell lines (HAP1 [Figure 2C] and HeLa R19 cells [Figure S3E]). "
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    ABSTRACT: Itraconazole (ITZ) is a well-known antifungal agent that also has anticancer activity. In this study, we identify ITZ as a broad-spectrum inhibitor of enteroviruses (e.g., poliovirus, coxsackievirus, enterovirus-71, rhinovirus). We demonstrate that ITZ inhibits viral RNA replication by targeting oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4). Consistently, OSW-1, a specific OSBP/ORP4 antagonist, also inhibits enterovirus replication. Knockdown of OSBP inhibits virus replication, whereas overexpression of OSBP or ORP4 counteracts the antiviral effects of ITZ and OSW-1. ITZ binds OSBP and inhibits its function, i.e., shuttling of cholesterol and phosphatidylinositol-4-phosphate between membranes, thereby likely perturbing the virus-induced membrane alterations essential for viral replication organelle formation. ITZ also inhibits hepatitis C virus replication, which also relies on OSBP. Together, these data implicate OSBP/ORP4 as molecular targets of ITZ and point to an essential role of OSBP/ORP4-mediated lipid exchange in virus replication that can be targeted by antiviral drugs. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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