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Bo Yu, Pei Xu, Zhen-Yang Zhao, Bo Long, Jiyang Cai, Yan Chen; Dissecting molecular pathways of mTORC1 signaling in the retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3443.
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Activity of the mechanistic target of rapamycin (mTOR) is controlled by upstream small GTPase protein Rheb, as well as its subcellular localization. Although a broad spectrum of downstream effectors exist for mTOR complex 1 (mTORC1), activated mTORC1 regulates specific substrates to accommodate with tissue- and cell-specific functions. The majority of literature studies on mTOR used transformed cancer cell lines whose signaling networks can be distinct from the retinal pigment epithelium (RPE). The purpose of this study is to explore RPE-specific pathways of mTOR, both in vitro and in vivo.
Major regulatory proteins of mTOR signaling, including TSC1, TSC2, Rheb, Rag and Ragulator complex, were down-regulated by specific siRNAs in cultured human fetal RPE cells or 143B osteosarcoma cells. Responses of mTORC1 and mTORC2 to nutrients and growth factors were monitored by measuring the phosphorylation status of S6 and Ser473 of Akt, respectively. To activate mTORC1 in vivo, mice were fed with a high fat, cholesterol rich (HF-C) diet which contained 20% fat (w/w) and 1.5% cholesterol for 1 month with or without administration of rapamycin. Activity of mTORC1 and subcellular location of mTOR-related proteins were analyzed in the isolated RPE/choroid tissue.
mTORC1 in the RPE and 143B cells both responded to nutrients and growth factors. However, regulation by the TSC1-TSC2 complex was different. In RPE cells, TSC1-TSC2 did not exert negative regulation on mTOR signaling. Instead, the presence of TSC complex was required for maximum activation of mTORC1. In vivo, HF-C diet selectively activated mTORC1 in the RPE, without affecting the Akt phosphorylation. Moreover, rapamycin regulated different sets of mTOR substrates in mice fed with HF-C diet as compared to animals on regular diet.
In contrast to transformed cells with strong mitogenic signaling, RPE cells have unique upstream regulatory mechanisms of mTORC1. There is a positive association between TSC1-TSC2 complex and mTORC1 activity in the RPE. How mTOR contributes to HF-C diet-induced RPE degeneration warrants further investigation.
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