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. 2008 Mar 14;29(5):541-51.
doi: 10.1016/j.molcel.2007.12.023.

Loss of the tuberous sclerosis complex tumor suppressors triggers the unfolded protein response to regulate insulin signaling and apoptosis

Umut Ozcan  1 , Lale Ozcan, Erkan Yilmaz, Katrin Düvel, Mustafa Sahin, Brendan D Manning, Gökhan S Hotamisligil
Affiliations

Loss of the tuberous sclerosis complex tumor suppressors triggers the unfolded protein response to regulate insulin signaling and apoptosis

Umut Ozcan et al. Mol Cell. .

Abstract

Mammalian target of rapamycin, mTOR, is a major sensor of nutrient and energy availability in the cell and regulates a variety of cellular processes, including growth, proliferation, and metabolism. Loss of the tuberous sclerosis complex genes (TSC1 or TSC2) leads to constitutive activation of mTOR and downstream signaling elements, resulting in the development of tumors, neurological disorders, and at the cellular level, severe insulin/IGF-1 resistance. Here, we show that loss of TSC1 or TSC2 in cell lines and mouse or human tumors causes endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). The resulting ER stress plays a significant role in the mTOR-mediated negative-feedback inhibition of insulin action and increases the vulnerability to apoptosis. These results demonstrate ER stress as a critical component of the pathologies associated with dysregulated mTOR activity and offer the possibility to exploit this mechanism for new therapeutic opportunities.

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Figures

Figure 1
Figure 1. Analysis of UPR and mTORC1 signaling pathways in Tsc1−/− and Tsc2−/− cells
(A) PERK (Thr980) phosphorylation, XBP-1 mRNA splicing and downstream elements of mTORC1 signaling; phosphorylation of S6K1 (Thr389), total S6K1 levels and S6 (Ser235/236) phosphorylation in the presence or absence of rapamycin (20 nM) for 24 hours in Tsc1−/− MEFs and WT controls (triplicates shown). (B) Expression levels of CHOP, GRP78, and XBP-1s mRNAs in Tsc1+/+ and Tsc1−/− cells treated either with rapamycin or vehicle. (C) UPR and mTOR signaling parameters in Tsc2−/− MEFs reconstituted with a retroviral control vector (+VEC) or with a retrovirus containing the WT TSC2 gene. (D) CHOP, GRP78, and XBP-1s mRNA levels in TSC2-deficient cells reconstituted with either vector or WT TSC2 upon treatment with rapamycin (20 nM) or vehicle, as above. (E) Time course of rapamycin’s effect on PERK phosphorylation in Tsc2−/− MEFs. Tsc2+/+ (WT) or Tsc2−/− cells were serum starved for 24 h in the presence of rapamycin (20 nM) for the duration indicated. Data are mean ± SEM of triplicates.
Figure 2
Figure 2. Increased UPR in tumors arising in TSC2+/− mice
(A) Hematoxylin & Eosin staining of the sections from the liver and (B) kidneys of Tsc2+/− mice at 12 months of age illustrating the presence of hemangiomas and adenomas, respectively. (C) Immunofluoresence staining of phospho-S6 (Ser235/236), phospho-IRE-1, phospho-PERK (Thr980) and phospho-eIF2α (ser51) in the liver hemangiomas of Tsc2+/− mice. (D) Immunofluoresence staining for phospho-S6 and GRP78 in the same cystic adenoma shown in panel B. (E) Detection of phospho-S6K1 (Thr389), total S6K1, phospho-PERK (Thr980), phospho-eIF2α (ser51) and total eIF2α levels by immunoblotting extracts from either adjacent normal kidney tissue (N) or kidney adenomas (T) removed from 20-months old Tsc2+/− mice treated with either rapamycin (10 mg/kg) or vehicle for 2 consecutive days. Mice were sacrificed and extracts were prepared 12 hours after the second dose. Representative samples are shown.
Figure 3
Figure 3. Analysis of UPR in a cortical tuber dissected from a 3 year-old patient with tuberous sclerosis
(A) Vimentin and phospho-S6 (ser235/236) staining of the tuber identify enlarged glial cells in which the mTORC1 pathway has been activated. Adjacent tissue contains many weakly-vimentin positive astroglia that do not contain phospho-S6 immunoreactivity. (B) Vimentin and phospho-eIF2α (ser51) staining in a tuber shows an enlarged cell that contains immunoreactivity for both glial marker vimentin and UPR marker phospho-eIF2α. (C) SMI-311 and phospho-eIF2α staining in a tuber shows an enlarged neuron, which contains immunoreactivity for the UPR marker phospho-eIF2α (ser51). Scale bar represents 50μm (D) SMI-311 and GRP78 staining in a tuber section shows several enlarged neurons that are immuno-reactive for the UPR marker GRP78. Scale bar represents 20μm.
Figure 4
Figure 4. Effect of PBA treatment on UPR in TSC1- and TSC2-deficient cells
(A) PERK (Thr980) phosphorylation after 4-PBA (10 mM) treatment for 24 hours in Tsc1−/− MEFs. Phospho-S6K-1 (Thr389), total S6K-1, and phospho-S6 (ser235/236) levels were analyzed with or without 4-PBA treatment in Tsc1−/− MEFs, shown in triplicate. (B) mRNA levels of CHOP, XBP-1s and GRP78 in Tsc1+/+ and in Tsc1−/− cells either in the presence or absence of 4-PBA (10mM) after 24 hours incubation. (C) PERK phosphorylation (Thr980), phospho-S6K-1 (Thr389), and total S6K-1, phospho-S6 (ser235/236) levels with or without 4-PBA treatment in Tsc2−/− (+VEC) MEFs and in their controls Tsc2−/− (+TSC2) cells. (D) CHOP, XBP-1s and GRP78 gene expression levels in WT (+TSC2) and Tsc2−/− (+VEC) cells treated with PBA (10mM) or PBS for 24 hours. Data are mean ± SEM of triplicates.
Figure 5
Figure 5. Role of UPR in development of insulin resistance in TSC1- and TSC2- deficient cells
(A) IP-kinase assay on JNK activity in Tsc2−/− cells reconstituted either with an empty retroviral vector or a TSC2 expressing vector after vehicle, rapamycin (20 nM) and 4-PBA (10 mM) treatment for 24 hours, shown in triplicate. (B–C) Analysis of insulin stimulated tyrosine phosphorylation of IRS-1 and IRS-2, and phosphorylation of Akt (serine 473) in Tsc1−/− (B) and Tsc2−/− (C) cells in the absence or presence of 4-PBA (10mM) for 24 hours. (D) IRS-1 tyrosine and Akt (serine 473) phosphorylations in Tsc2−/− (+TSC2) and Tsc2−/− (+VEC) MEFs after 4-PBA treatment. (E) Ubiquitination of IRS-1 after 8 hours of Thapsigargin (300 nM) or Tun (2 μg/ml) treatment in the presence of epoximicin (1 μg/ml) (F) IRS-1 protein levels in the presence of cycloheximide (100 μg/ml) after 20 hours of 4-PBA (10 mM) pre-treatment of Tsc1−/− cells. (G). Decreased ER stress and recovery of insulin-stimulated IRS-1 and Akt phosphorylation in Tsc2−/− MEFs by expression of exogenous spliced XBP-1 (sXBP-1). sXBP-1 is introduced into the cells by an adenoviral expression system and the bottom panel shows the protein levels obtained in the cells. Phosphorylation of PERK is examined to monitor ER stress.
Figure 6
Figure 6. TSC1- and TSC2-deficiency increases vulnerability to ER stress induced apoptosis
(A) TSC1- and TSC2-deficient cells and their corresponding controls were incubated overnight in 2% FBS-containing medium and then treated with thapsigargin (300nM) for 6 hours and apoptosis levels were analyzed by ELISA (B) Tsc2−/− cells reconstituted either with TSC2 or with a control vector were stimulated with tunicamycin (2 μg/ml) for 14 hours in the presence of rapamycin (20nM) or DMSO as control. Immunoblotting was performed to analyze the levels of cleaved caspase-3 and cleaved PARP. (C) Tsc2−/− cells reconstituted with vector or TSC2 were starved of glucose for 16 hours in the presence or absence of 4-PBA (10 mM) or rapamycin (20 nM). (D). Induction of apoptosis in Tsc-deficient tumors in vivo by treatment with thapsigargin. Tsc-deficient tumor and surrounding normal tissues were sectioned following thapsigargin treatment and stained with H&E and Tunel to detect apoptotic cells. (E). A model of the interactions between TSC function and insulin resistance. TSC-deficiency results in increased mTOR activity which triggers uncontrolled protein synthesis leading to development ER stress and through this mechanism, insulin resistance. In this setting, ER stress also sensitizes cells to apoptosis. Data are mean ± SEM of triplicates.
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