The nutrient-responsive transcription factor TFE3 promotes autophagy, lysosomal biogenesis, and clearance of cellular debris

Abstract

The discovery of a gene network regulating lysosomal biogenesis and its transcriptional regulator transcription factor EB (TFEB) revealed that cells monitor lysosomal function and respond to degradation requirements and environmental cues. We report the identification of transcription factor E3 (TFE3) as another regulator of lysosomal homeostasis that induced expression of genes encoding proteins involved in autophagy and lysosomal biogenesis in ARPE-19 cells in response to starvation and lysosomal stress. We found that in nutrient-replete cells, TFE3 was recruited to lysosomes through interaction with active Rag guanosine triphosphatases (GTPases) and exhibited mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1)-dependent phosphorylation. Phosphorylated TFE3 was retained in the cytosol through its interaction with the cytosolic chaperone 14-3-3. After starvation, TFE3 rapidly translocated to the nucleus and bound to the CLEAR elements present in the promoter region of many lysosomal genes, thereby inducing lysosomal biogenesis. Depletion of endogenous TFE3 entirely abolished the response of ARPE-19 cells to starvation, suggesting that TFE3 plays a critical role in nutrient sensing and regulation of energy metabolism. Furthermore, overexpression of TFE3 triggered lysosomal exocytosis and resulted in efficient cellular clearance in a cellular model of a lysosomal storage disorder, Pompe disease, thus identifying TFE3 as a potential therapeutic target for the treatment of lysosomal disorders.

Fig. 1. TFE3 redistributes from the cytosol to the nucleus upon mTORC1 inactivation, nutrient deprivation, or lysosomal stress

(A) Immunofluorescence confocal microscopy analysis of the subcellular distribution of TFE3-MYC in ARPE-19 cells incubated with Torin-1, starved in serum and amino acid-free medium, or exposed to Chloroquine for 1 hour. Cells were double stained with antibodies against TFE3 (a dilution of 1:7000 was used to detect recombinant TFE3) and LAMP1. Insets show a 2.5-fold magnification of the indicated region. Scale bars, 10 μm. Data are representative of 3 independent experiments and over 90% of cells exhibited the phenotypes shown. (B) Immunofluorescence confocal microscopy analysis of the subcellular distribution of endogenous TFE3 in ARPE-19 cells exposed to the same condition as those indicated in (A). Cells were stained with antibodies against TFE3 (a dilution of 1:200 was used to detect endogenous TFE3). Scale bars, 10 μm. Data are representative of 3 independent experiments and over 90% of cells exhibited the phenotypes shown. (C) Top: Immunoblotting showing changes on TFE3 electrophoretic mobility after 2 h incubation with Torin-1. Bottom: Immunoblotting analysis of the subcellular distribution of endogenous TFE3 in ARPE-19 cells incubated with DMSO or Torin-1 for 2 hours, or starved in a medium without serum and amino acids for 20 hours. The subcellular fractions were probed with antibodies against TFE3, LAMP1 (lysosomal membrane marker), and Histone H3 (nuclear marker). (D) Immunoblotting analysis of coimmunoprecipitated 14-3-3 with TFE3-MYC in ARPE-19 cells treated with DMSO (control) or Torin-1 for 1hour. Protein bands were detected with antibodies against MYC (used to detect TFE3-MYC) and 14-3-3. Data in C and D are representative of 3 independent experiments.

Fig. 2. TFE3 interacts with active Rag heterodimers

(A) Schematic representation of comparison between TFEB and TFE3. RBD: Rag-binding domain, AD: Activation domain, NIS: Nuclear import signal, B: Basic residues, HLH: Helix-loop-helix, LZ: Leucine-zipper, PRO-rich: Proline rich. Residues proven to be essential in the RBD (S112R113), 14-3-3 binding site (S321), and NIS (R356R357R358) are colored. (B and C) Immunoblotting analysis of TFE3-MYC (B) or endogenous TFE3 (C) affinity purified with glutathione S-transferase (GST) fused to C-terminus of active Rag heterodimers (RagB_GTP/RagD_GDP) in ARPE-19 cells. The affinity-purified materials were probed with antibodies against GST, MYC, and TFE3 (used to detect Rag proteins, TFE3-MYC, and endogenous TFE3, respectively). Data are representative of 3 independent experiments. (D) Immunofluorescence confocal microscopy analysis of the subcellular distribution of endogenous TFE3 in ARPE-19 cells overexpressing either active or inactive RagB/D heterodimers. Cells were double stained with antibodies against TFE3 and GST (used to detect endogenous TFE3 or Rag proteins, respectively). Asterisks indicate distribution of endogenous TFE3 in non-transfected cells. Scale bars, 10 μm Data are representative of 3 independent experiments and over 90% of cells positive for the RagB/D heterodimers exhibited the phenotypes shown. (E) Immunofluorescence confocal microscopy analysis of the subcellular distribution of endogenous TFE3 in control or Rag-depleted ARPE-19 cells upon incubation with Torin-1 for 1 hour. Cells were double stained with antibodies against TFE3 and LAMP1. Scale bars, 10 μm. Data are representative of 3 independent experiments and over 90% of cells exhibited the phenotypes shown.

Fig. 3. Folliculin is recruited to lysosomes and upregulated by TFE3 during starvation

(A) ARPE-19 cells were transfected with siRNA duplexes to FLCN or non-target siRNA for 72 hours. Cells were fixed and the nuclear localization was assessed by immunofluorescence microscopy using an antibody that recognizes TFE3. Data are shown as means ± SD and represent 643 cells (siRNA Non-target) and 663 cells (siRNA FLCN) counted in 3 independent experiments. The data were analyzed using paired t test (***, p=0.003). (B) ARPE-19 cells were starved in serum-and amino acid-free medium for 2 hours (Starvation) or starved and re-fed (Re-feed) in complete medium for 30 minutes. Cells were fixed and FLCN subcellular localization was analyzed by immunofluorescence confocal microscopy using antibodies to FLCN and LAMP1. Insets represent a two-fold magnification of the indicated region. Scale bars, 10 μm. Data are representative of 3 independent experiments and over 90% of cells exhibited the phenotypes shown. (C) ARPE-19 cells were transfected with either active or inactive RagB/D heterodimers. After 12 hours, cells were double stained with antibodies against HA (used to detect Rag proteins) and FLCN. Scale bars, 10 μm. Arrows indicate cells transfected with inactive RagB/D heterodimers. Data are representative of 3 independent experiments and over 90% of HA-positive cells exhibited the phenotypes shown. (D) ARPE-19 were nucleofected with the indicated GST-Rag-expressing plasmids. Twenty hours later, cells were lysed, and RagB/D heterodimers were pulled down using glutathione-Sepharose beads. Proteins bound to the beads were analyzed by immunoblotting with antibodies against GST (used to detect Rag proteins), TFE3 and FLCN. Data are representative of 3 independent experiments (E) ARPE-19 cells were transfected with siRNA duplexes to TFE3 or non-target siRNA for 48 hours. Cells were then either kept in complete medium (control) or starved in serum- and amino acid-free medium for 24 hours. A representative blot is shown along with quantification of three independent experiments plotted as the ratio of FLCN to actin is expressed as fold increase relative to the control Non-target siRNA condition. The data were analyzed using one-way ANOVA (*, p ≤ 0.05; and n.s.: not significant starved versus control for each siRNA condition). (F) ARPE-19 cells were infected with either adenovirus expressing Null (Ad. Null) or TFE3-MYC (Ad. TFE3) for 48 hours. Cells were then lysed and the lysates were analyzed by immunoblotting using antibodies to detect FLCN, MYC (TFE3), and actin. Data are representative of 3 independent experiments (G) ARPE-19 cells were infected with either adenovirus expressing Null, TFE3-MYC, or TFEB-FLAG for 48 hours and RNA was extracted. mRNA transcript abundance was assessed by Relative Quantitative Real Time PCR using specific primers for the indicated genes from three independent experiments. The data were analyzed using one-way ANOVA and are shown as means ± SD (*, p ≤ 0.05; **, p ≤ 0.01; versus adenovirus Null-infected cells).

Fig. 4. TFE3 overexpression induces the transcription of autophagy-related genes

(A) ARPE-19 cells were infected with Null-, MITF1-, TFEB-, or TFE3-expressing adenoviruses for 48 hours and total RNA was extracted. mRNA transcript abundance was assessed using the Human Autophagy RT² Profiler PCR Array. Represented genes exhibited at least 2-fold increase in cells overexpressing TFE3. Values are means ± SD of 3 independent Profiler experiments. The data were analyzed using one-way ANOVA (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ns: not significant versus Null-infected cells). (B) Immunoblotting analysis of ARPE-19 cells overexpressing adenovirus Null, TFEB-FLAG, MITF1-FLAG or TFE3-MYC. Proteins were detected with antibodies against FLAG (used to detect TFEB-FLAG and MITF-FLAG), MYC (used to detect TFE3-MYC), LC3, and Actin. Data are representative of 3 independent experiments. (C) Quantification of LC3_I and LC3_II protein bands as shown in B. Data were normalized to actin. Bars represent ratio of LC3_II/LC3_I expressed as fold increase of the ratio from cells infected with adenovirus Null. Values represent the average ± range of 2 independent experiments. (D) ARPE-19 cells were infected as in A with the indicated adenoviruses, fixed, and then stained with LC3 for detection of autophagosomes. Data are representative of 3 independent experiments and over 80% of cells exhibited the phenotypes shown.

Fig. 5. TFE3 overexpression induces transcription of lysosomal genes through the CLEAR element

(A) ARPE-19 cells were infected with Null-, MITF1-, TFEB-, or TFE3-expressing adenoviruses for 48 hours and RNA was extracted. mRNA transcript abundance was assessed by qRT-PCR using specific primers for the indicated genes. Data are presented as means ± SD of three independent experiments. The data were analyzed using one-way ANOVA(*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ns: not significant versus Null-infected cells) (B) Immunofluorescence confocal microscopy analysis of LAMP1 in ARPE-19 cells infected with Null- or TFE3-MYC-expressing adenoviruses. Cells were double stained with antibodies against MYC (to detect recombinant TFE3) and LAMP1. Scale bars, 10 μm. Quantification of LAMP1-positive puncta shown for 25 cells per condition in 3 independent experiments. Data are presented as means ± SD (C) Luciferase activity measured in cells coexpressing the MCOLN1 promoter-luciferase reporter constructs with Null, TFEB-WT or TFE3-WT. Wild-type contains all three CLEAR elements, ΔClear 1 lacks the CLEAR element at position −170, ΔClear 2 lacks the CLEAR element at position −143, ΔClear 3 lacks the CLEAR element at position −123, ΔClear 1+2 lacks the CLEAR elements at position −170 and −123, ΔClear 1+3 lacks the CLEAR elements at position −170 and −123, ΔClear 2+3 lacks the CLEAR elements at position −143 and −123, ΔClear 1+2+3 lacks all three CLEAR elements. Bars represent luciferase activity expressed as fold increase versus cells infected with adenovirus Null. Values are means ± SD of three independent experiments. The data were analyzed using one-way ANOVA (***, p ≤ 0.001 versus Null-infected cells).

Fig. 6. TFE3 promotes expression of lysosomal genes independently of TFEB

(A) Immunoblotting analysis of control (Null) or TFEB-depleted HeLa cells infected with Null- or TFE3-MYC-expressing adenoviruses for 48 hours. Protein bands were detected with antibodies against TFEB and MYC (used to detect endogenous TFEB and TFE3-MYC, respectively), and Actin. Data are representative of 3 independent experiments. (B) qRT-PCR analysis of lysosomal genes from HeLa cells treated as indicated in (A). Total RNAs were extracted and mRNA transcript abundance was assessed using specific primers for the indicated genes. Data were normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Bars represent fold change of the ratio to shRNA non-target cells infected with adenovirus Null. Values are presented as average ± range of 2 independent experiments. (C) Immunoblotting analysis of ARPE-19 cells treated with siRNA to TFEB, TFE3, or TFEB and TFE3 (TFEB+TFE3) under control or starvation conditions. Proteins were detected with antibodies against TFEB, TFE3, and Actin. Note that the immunoblot for endogenous TFEB required a much longer exposure time due to the very low abundance of TFEB in ARPE-19 cells compared to the abundance of endogenous TFE3. Data are representative of 3 independent experiments (D) qRT-PCR analysis of lysosomal- or autophagy-related genes from ARPE-19 cells treated as indicated in (C). Total RNAs were extracted and mRNA transcript abundance was assessed using specific primers for the indicated genes. Bars represent fold change of the ratio to siRNA non-target cells in control condition. Values are means ± SD of 3 independent experiments. The data were analyzed using one-way ANOVA (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 versus siRNA non-target control cells).

Fig. 7. TFE3 overexpression triggers lysosomal exocytosis

(A) Immunofluorescence confocal microscopy analysis of endogenous MCOLN1 in ARPE-19 cells infected with Null adenovirus or adenovirus expressing TFEB-FLAG, MITF1-FLAG, or TFE3-MYC for 40 hours. Cells were double stained with antibodies against MCOLN1 and LAMP1. Scale bars, 10 μm. Data are representative of 3 independent experiments and over 90% of cells exhibited the phenotypes shown. (B) Immunoblotting analysis of ARPE-19 cells overexpressing adenovirus Null, TFEB-FLAG, MITF1-FLAG, or TFE3-MYC. Proteins were detected with antibodies against MCOLN1, MYC (to detect TFE3), FLAG (to detect TFEB and MITF1), and Actin. Data are representative of 3 independent experiments. (C) Acid phosphatase secretion analysis in HeLa cells infected with adenoviruses expressing the indicated proteins. Bars represent the secreted acid phosphatase activity as a percentage of the total acid phosphatase activity. Values are means ± SD of 3 independent experiments. The data were analyzed using one-way ANOVA (***, p ≤ 0.001; ns: not significant versus Null-infected cells). (D) Immunofluorescence confocal microscopy analysis of LAMP1 present at the cell surface of ARPE-19 cells overexpressing TFEB-FLAG, MITF1-FLAG, or TFE3-MYC. Cells were double stained with antibodies against FLAG and MYC (used to detect TFEB-FLAG, MITF-FLAG, and TFE3-MYC, respectively) and LAMP1. Scale bars, 10 μm. Data are representative of 3 independent experiments and over 90% of cells exhibited the phenotypes shown.

Fig. 8. TFE3 promotes clearance of enlarged lysosomes and reduces glycogen load in PD myotubes

(A) Confocal microscopy images of PD myotubes (clone 6) infected with adenovirus expressing TFE3-MYC for 24, 48, or 72 hours. Ad. TFE3 was added on day 4, 5, and 6 after the differentiation began (see Materials and Methods). The cells were then fixed on day 7 and stained with LAMP-1 (lysosomes; red) and MYC (TFE3; green) antibodies. Nuclei are stained with Hoechst (blue). Insets show a 1.5 fold magnification of the indicated region. Graphical representation of the quantification of the data in A. The bars represent the number of enlarged lysosomes (>2 μm) per μm² of the myotube in uninfected cells and cells infected for 48 or 72 hours with adenovirus expressing TFE3-MYC. Values are means ± SD of 3 independent experiments. The data were analyzed using one-way ANOVA (**, p ≤ 0.01). (B) LysoTracker staining of live PD cells infected with the Null or TFE3-MYC-expressing adenovirus. Adenovirus was added to the myotubes for 72 hours on day 7 in differentiation medium. Insets show a 1.7 fold magnification of the indicated region. Data are representative of 3 independent experiments and over 90% of cells exhibited the phenotypes shown. (C) Confocal microscopy images of live noninfected PD myotubes (Top) or PD myotubes infected for 48 hours with adenovirus expressing TFE3-MYC (Bottom). The cells were incubated with the fluorescent glucose (2-NBDG; green), washed, and analyzed by confocal microscopy. Scale bars, 10 μm for all panels. Data are representative of 3 independent experiments and over 90% of cells exhibited the phenotypes shown. (D) Accumulation of TFE3 in the nuclei in myotubes infected with TFE3-MYC for 48 or 72 hours. TFE3 was detected with antibody recognizing MYC (red). (E) Surface LAMP assay of PD myotubes infected with Null- or TFE3-MYC-expressing adenovirus. Confocal microscopy images show LAMP1 staining (red) on plasma membrane in TFE3-treated cells (bottom; arrowheads) but not in noninfected cells (top). Nonpermeabilized cells were incubated with antibody recognizing LAMP1 at 4°C for 40 min, followed by fixation and staining with secondary antibody. Data are representative of 3 independent experiments and over 70% of cells exhibited the phenotypes shown. Bar: 10 μm for all panels.