Figure 6. Mechanistic target of rapamycin complex 1 (mTORC1) regulates cystine uptake through activating transcription factor 4 (ATF4) and its target SLC7A11.
(A) Schematic of transporter xCT, encoded by Slc7a11, which heterodimerizes with SLC3A2 to serve as a cystine (Cys2)/glutamate anti-porter. Cystine is reduced to cysteine (Cys), which is essential for glutathione synthesis. Cysteine transport is mediated by neutral amino acid trasporters distinct from xCT. The targets of erastin and buthionine-sulfoximine, two compounds used in this study, are also depicted. (B, C) qPCR analysis of Slc7a11 (B) or Slc3a2 (C) in serum-deprived Tsc2-/- mouse embryo fibroblasts (MEFs) (wild-type [WT]) and Tsc2-/- Atf4-/- MEFs with stable expression of cDNAs encoding GFP (control), ATF4 lacking its 5′-UTR, or a DNABD mutant (DBD) of this ATF4 treated with vehicle or rapamycin (20 nM, 16 hr). Expression relative to WT vehicle-treated cells is graphed as the log2 mean ± SD from a representative experiment with three biological replicates (n = 3). (D–F) Representative immunoblots of serum-deprived Tsc2-/- MEFs (D), insulin-stimulated (500 nM, 24 hr) WT MEFs (E), or serum-deprived LNCaP cells (F) treated 24 hr (D, E) or 16 hr (F) with vehicle, rapamycin (20 nM), or Torin1 (250 nM), shown with biological duplicates. The SLC7A11 antibody is validated for use in MEFs in Figure 6—figure supplement 1A, corresponding immunoblots quantified in Figure 6—figure supplement 1B, C, F. Effects of ATF4 knockdown and rapamycin on SLC7A11 transcripts in LNCaP and PC3 cells are shown in Figure 6—figure supplement 1D, E, and corresponding immunoblots and protein quantification are provided in Figure 6—figure supplement 1G, H. (G, H) Representative growth curves of Tsc2-/- Atf4-/- MEFs with stable expression of GFP, ATF4, or ATF4DBD grown in 10% dialyzed fetal bovine serum (FBS) with Dulbecco’s Modified Eagle’s Medium (DMEM) (G) or DMEM supplemented with cysteine alone (Cys, 1 mM), nonessential amino acids (100 μM each) lacking cysteine (NEAA-Cys), or nonessential amino acids plus either cysteine (1 mM, NEAA+Cys), or cystine (0.5 mM, NEAA+Cys2) (H). Mean cell numbers ± SD relative to day 0 are graphed from three biological replicates (n = 3). (I) Cell death of Tsc2-/- Atf4-/- MEFs with stable expression of cDNAs encoding GFP, ATF4, or SLC7A11 cultured in DMEM with 10% dialyzed FBS was quantified by annexin V and propidium iodide (PI) staining after 72 hr and graphed as the mean percentage of total cells ± SD from three biological replicates (n = 3). (J) Cystine uptake in serum-deprived Tsc2-/- MEFs treated with vehicle, rapamycin (20 nM), or erastin (10 μM) for 16 hr is graphed as the mean ± SEM radiolabel incorporation from C14-cystine over the final 10 min relative to vehicle-treated cells from two independent experiments, with three biological replicates each (n = 6). The effect of mTOR inhibitors on cystine uptake in littermate-derived Rictor+/+ and Rictor-/- MEFs, and corresponding immunoblots, is shown in Figure 6—figure supplement 1I, J. (K) Cystine uptake in serum-deprived WT and Atf4-/- MEFs pretreated 30 min with vehicle or rapamycin (20 nM) prior to insulin stimulation (500 nM, 24 hr) or treated with erastin (10 μM, 30 min) was assayed and graphed as in (J) relative to vehicle-treated WT cells with data from three independent experiments, with three biological replicates each (n = 9). (L) Cystine uptake in serum-deprived Tsc2-/- MEFs (WT) andTsc2-/- Atf4-/- MEFs with stable expression of cDNAs encoding GFP or ATF4 treated with vehicle or rapamycin (20 nM) for 16 hr was assayed and graphed as in (J) relative to vehicle-treated WT cells with data from two independent experiments, with three biological replicates each (n = 6). (B–I) are representative of at least two independent experiments. *p<0.05, **p<0.01, ***p<0.001, ns = not significant, calculated in (B, C, I, J, K, L) via one-way analysis of variance with Holm–Sidak method for multiple comparisons and in (G, H) via unpaired two-tailed t-test. For (I), the sum of annexin V+/PI-, annexin V-/PI+, and annexin V+/PI+ populations were used for comparisons to the annexin V-/P- population.
Figure 6—figure supplement 1. Supplemental data supporting Figure 6.
