Research ArticleFibrosis

mTORC1 amplifies the ATF4-dependent de novo serine-glycine pathway to supply glycine during TGF-β1–induced collagen biosynthesis

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Science Signaling  21 May 2019:
Vol. 12, Issue 582, eaav3048
DOI: 10.1126/scisignal.aav3048
  • Fig. 1 Identification of a rapamycin-insensitive, mTOR-dependent serine-glycine biosynthetic signature during TGF-β1–induced collagen deposition.

    (A) Plot showing scaled gene expression intensities from the rapamycin-insensitive mTOR module eigengene as calculated by WGCNA. The module eigengene is defined as the first principal component of the genes contained within the module and is representative of the gene expression profiles in the module. All expression values have been z-transformed, and signals that are negatively correlated to the module eigengene have been inverted for plotting (n = 4 independent experiments). (B) Bar plot showing the top 10 most significantly enriched pathways for the genes in the rapamycin-insensitive mTOR module. The serine-glycine biosynthesis pathway was most enriched (P = 5.45 ×10−5). (C) Heat map representing the genes from the top 20 most enriched pathways in the rapamycin-insensitive mTOR module, listed in order of the most to the least statistically significant. Genes that map to more than one pathway only appear for the pathway with the most significant P value. Scaled counts were used to generate the heat map, where darker red indicates higher number of counts. The black arrowheads indicate the genes belonging to the glycine metabolism pathway, and the clear arrowhead indicates SLC2A1 (n = 4 independent experiments). (D) Confluent primary human lung fibroblasts (pHLFs) were stimulated with media alone or media plus TGF-β1, and extracts were immunoblotted for the indicated proteins. Representative data are shown (n = 3 independent experiments). (E) Confluent pHLFs were preincubated with media plus vehicle [dimethyl sulfoxide (DMSO)] or AZD8055 and stimulated for 48 hours with or without TGF-β1. Collagen I deposition was assessed by high-content imaging. Half-maximal inhibitory concentration (IC50) value was calculated using four-parameter nonlinear regression. Each data point shown is the mean ± SEM of the fold change to baseline of three technical replicates per condition. Data are representative of three independent experiments. (F) Immunofluorescence staining showing collagen I deposition in pHLFs treated as in (E). Scale bar, 100 μm. DAPI, 4′,6-diamidino-2-phenylindole. (G) Confluent pHLFs were preincubated with media plus vehicle or rapamycin and stimulated for 48 hours with or without TGF-β1. Collagen I deposition was assessed by high-content imaging. Each data point shown is the mean ± SEM of the fold change relative to baseline of three technical replicates per condition. Data are representative of three independent experiments.

  • Fig. 2 TGF-β1 amplifies the serine-glycine biosynthesis pathway in an mTOR-dependent manner.

    Confluent pHLFs were incubated in media alone or media plus TGF-β1 with AZD8055 or vehicle control (DMSO) for 24 hours. (A to D) Quantification of the relative abundance of PHGDH (A), PSAT1 (B), PSPH (C), and SHMT2 (D) mRNAs by real-time quantitative polymerase chain reaction (RT-qPCR). Data are presented as means ± SEM from three technical replicates per condition and representative of three independent experiments. (E and F) Immunoblots of protein lysates and densitometric quantification of PHGDH (E) and PSPH (F). Data are representative of three independent experiments with three technical replicates per condition. Differences between groups were evaluated by two-way analysis of variance (ANOVA) test with Tukey post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. a.u., arbitrary units.

  • Fig. 3 mTOR plays a key role in promoting ATF4 protein production.

    (A) OCN analysis of the RNA-seq data revealed a cluster of transcription factors associated with the serine-glycine module of enriched mRNAs. (B) Confluent pHLFs were incubated with or without TGF-β1, and the relative abundance of ATF4 mRNA over time was measured by RT-qPCR. (C) Immunoblots and densitometric quantification of ATF4 protein abundance in pHLF lysates over time after TGF-β1 stimulation. The immunoblot shows ATF4 at 8 hours after TGF-β1 addition. (D) Relative abundance of ATF4 mRNA measured 24 hours after TGF-β1 addition. (E) Immunoblot and densitometric quantification of ATF4 abundance at 24 hours after TGF-β1 addition. (F) Immunoblot and densitometric quantification of ATF4 abundance at the indicated times after TGF-β1 addition. (G) Confluent pHLFs were transfected with scrambled control siRNA (small interfering RNA) (siCTRL) or Smad3 siRNA (siSMAD3) and incubated with or without TGF-β1. Relative abundance of ATF4 mRNA at 24 hours was measured by RT-qPCR. (H and I) ATF4 immunoblots and densitometric quantification for samples treated as in (G) analyzed at 8 hours (H) and 24 hours (I). (J) pHLFs were pretreated with TGF-β1 for 13 hours before treatment with lactimidomycin (LTM) plus either vehicle (DMSO) or AZD8055. Lysates were harvested at indicated times after treatment, and ATF4 abundance was measured by immunoblotting and densitometric quantification. (K) pHLFs were modified by CRISPR-Cas9 gene editing of RPTOR or RICTOR and stimulated with TGF-β1. Immunoblot for ATF4 and densitometric quantification were performed at 24 hours. (L) pHLFs expressing a 4E-BP1-4A dominant-negative phospho-mutant were induced with doxycycline or media alone for 24 hours before TGF-β1 stimulation. Immunoblotting for ATF4 and densitometric quantification were performed at 18 hours after TGF-β1 addition. All data are expressed as means ± SEM from three technical replicates per condition and representative of three independent experiments. Differences between groups were evaluated by two-way ANOVA test with Tukey post hoc test (B to I and L), repeated-measures two-way ANOVA (J), or one-way ANOVA with Tukey post hoc test (K). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 4 ATF4 colocalizes with α-SMA–positive myofibroblasts within IPF fibrotic foci.

    (A to C) Immunofluoresence showing ATF4 (A, green), α-SMA (B, red), and the overlay of ATF4 and α-SMA (C, yellow) in a representative IPF fibrotic focus. The arrow indicates myofibroblasts within the fibrotic focus, and the arrowhead points to the hyperplastic epithelium. (D) Overlay of ATF4 and α-SMA in non-IPF lung tissue. (E to G) Corresponding high-magnification images of ATF4 (E), α-SMA (F), and the overlay (G) in myofibroblasts within the fibrotic focus indicated by the arrow in (A). (H) Mid-level noncomposite confocal overlay image, of the same capture region in (G), showing nuclear localization of ATF4 (green) in an α-SMA–positive myofibroblast cell. All images were counterstained with DAPI (blue). Scale bars, 50 μm (A to D) and 25 μm (E to H). n = 3 patients with IPF; n = 2 control subjects. Representative images are shown.

  • Fig. 5 ATF4-dependent modulation of the serine-glycine pathway is critical for TGF-β1–induced collagen deposition.

    (A) Confluent pHLFs were incubated with media plus TGF-β1 or media alone for 8 or 24 hours before cell lysis and separation into cytoplasmic (Cyto), nuclear (Nuc), and chromatin (Chrom) fractions that were immunoblotted for ATF4 and histone H3. (B to F) Confluent pHLFs were transfected with ATF4 siRNA (siATF4) or scrambled control (siCTRL) before exposure to media plus TGF-β1 or media alone. The relative abundances of ATF4, PHGDH, PSAT1, PSPH, and SHMT2 mRNAs were measured after 24 hours by RT-qPCR. (G) Representative immunoblots of protein lysates treated as indicated in (B) to (F). Data are representative of three independent experiments with three technical replicates per condition. (H) Confluent pHLFs were transfected with ATF4 siRNA (siATF4) or scrambled control (siCTRL) before exposure to media plus TGF-β1 or media alone. Collagen deposition was assayed by high-content imaging after 48 hours. Each data point shown is the mean ± SEM of the fold change relative to baseline of three to four technical replicates per condition, and data are representative of three independent experiments. (I) Representative immunofluorescence images showing collagen production by cells in (H). Scale bar, 100 μm. (J) Confluent wild-type and ATF4−/− pHLFs were exposed to media plus TGF-β1 or media alone. Collagen deposition was assayed by high-content imaging after 48 hours. Each data point shown is the mean ± SEM of the fold change relative to baseline of three technical replicates per condition, and data are representative of three independent experiments. Differences between groups were evaluated by two-way (B to F, H, and J) ANOVA test with Tukey post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 6 mTOR amplifies glucose metabolism during TGF-β1–induced collagen synthesis through an ATF4-dependent mechanism.

    (A) Confluent pHLFs were exposed to media plus TGF-β1 or media only for 24 hours. The area under the curve (AUC) of lactate relative to that of glucose in cell supernatants was measured by nuclear magnetic resonance (NMR) spectroscopy at the indicated time points. Data are representative of three independent experiments with three technical replicates per condition. ppm, parts per million. (B and C) Confluent pHLFs were exposed to media plus TGF-β1 or media only for 24 hours. ECAR and OCR were measured using the Seahorse XFe96 assay. Data are representative of three independent experiments with 46 technical replicates per condition. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. (D and E) Confluent pHLFs were incubated in glucose-depleted media (D) or preincubated with rotenone and antimycin A (E) before stimulation for 48 hours with or without TGF-β1. Collagen deposition was assessed by high-content imaging. Each data point shown is the mean ± SEM of the fold change relative to baseline of three technical replicates per condition. Data are representative of three independent experiments. (F) Confluent pHLFs were preincubated with AZD8055 or vehicle control (DMSO) before exposure to media plus TGF-β1 or media alone. The AUC of the lactate peak relative to the AUC of the glucose peak in cell supernatant was measured by NMR spectroscopy after 24 hours. Data are representative of three independent experiments with three technical replicates per condition. (G) The ECAR was assayed by Seahorse XF96e after 24 hours with or without TGF-β1 stimulation Data are representative of three independent experiments with three technical replicates per condition. (H to K) Confluent pHLFs were preincubated with AZD8055 or vehicle control before stimulation with TGF-β1 or media alone. Relative mRNA abundances of PFKFB3 at 3 hours after TGF-β1 (H), LDHA (I), and SLC2A1 (J) at 24 hours after TGF-β1 was measured by RT-qPCR. Cell extracts were subjected to immunoblotting and densitometric quantification for GLUT1 (K) (n = 3 independent experiments). (L) Confluent pHLFs were transfected with ATF4 siRNA (siATF4) or scrambled control (siCTRL) and then exposed to media plus TGF-β1 or media only. Relative abundance of SLC2A1 mRNA at 24 hours was measured by RT-qPCR. Data are representative of three independent experiments with three technical replicates per condition. (M) Representative immunoblot and densitometric quantification of GLUT1 in lysates from pHLFs treated as in (L), and data are representative of three independent experiments with three technical replicates per condition. (N) Confluent pHLFs were transfected with PHGDH siRNA (siPHGDH) or scrambled control (siCTRL) and exposed to media plus TGF-β1 or media only for 48 hours. Collagen deposition was assessed by high-content imaging. Each data point shown is the mean ± SEM of the fold change relative to baseline of three technical replicates per condition and is representative of three independent experiments. (O) pHLFs were treated with the PHGDH inhibitor NCT-503 or vehicle control and stimulated with or without TGF-β1 for 48 hours. Collagen deposition was assessed by high-content imaging. Each data point represents the mean ± SEM of the fold change relative to baseline of five technical replicates per condition and is representative of three independent experiments. Differences between groups were evaluated by unpaired t test (B) or two-way ANOVA test with Tukey post hoc test (A and C to O). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 7 mTOR promotes glycine biosynthesis from glucose to support TGF-β1–induced collagen synthesis.

    (A) Confluent pHLFs were deprived of glucose for 24 hours in the presence or absence of glycine, followed by TGF-β1 stimulation for 48 hours before assessment of collagen deposition by high-content imaging. Each data point shown is the mean ± SEM of the fold change relative to baseline of three technical replicates per condition. Data are representative of three independent experiments. (B) Confluent pHLFs were incubated with AZD8055, supplemented 30 min later with or without glycine, and then incubated with or without TGF-β1 for 48 hours before assessment of collagen deposition by high-content imaging. Each data point shown is the mean ± SEM of the fold change relative to baseline of three technical replicates per condition. Data are representative of three independent experiments. (C) Confluent pHLFs were incubated with AZD8055 or vehicle (DMSO) and exposed to media plus TGF-β1 or media alone in the presence of glucose for 48 hours. Collagen α1(I) was isolated by immunoprecipitation and immunoblotted. (D) Confluent pHLFs were incubated with AZD8055 or vehicle and then exposed to media plus TGF-β1 or media alone in the presence of U-14C-glucose for 48 hours. U-14C-glucose incorporation into immunoprecipitated collagen α1(I) was assessed by scintillation counting. Data are presented as means ± SEM from three technical replicates per condition and are representative of three independent experiments. (E) Confluent pHLFs were incubated with AZD8055 or vehicle and then stimulated with TGF-β1 in the presence of U-14C-glycine for 48 hours. U-14C-glycine incorporation into immunoprecipitated collagen α1(I) was assessed by scintillation counting and expressed relative to immunoprecipitated collagen α1(I) abundance quantified in a parallel immunoblot. Data are presented as means ± SEM from three technical replicates per condition and are representative of three independent experiments. Differences between groups were evaluated by unpaired t test (E) or one-way (A, B, and D) ANOVA test with Tukey post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 8 Model for ATF4-mediated metabolic and biosynthetic network reprogramming to support enhanced collagen biosynthesis in TGF-β1–stimulated myofibroblasts.

    TGF-β1–induced activation of the TGF-β receptor complex leads to a Smad3-dependent increase in ATF4 mRNA abundance and mTOR activation. Activated mTORC1–4E-BP1 signaling, in turn, promotes ATF4 protein production through a translational mechanism. ATF4 subsequently promotes the transcription of key serine-glycine pathway genes and SLC2A1 and, therefore, an increase in the abundance of the SLC2A1 gene product, GLUT1. The serine-glycine biosynthesis enzymes and GLUT1 act together to promote glucose-derived glycine biosynthesis to support enhanced collagen synthesis rates in activated myofibroblasts. G6P, glucose 6-phosphate; 3-PG, 3-phosphoglycerate; 3-PHP, 3-phosphohydroxypyruvate; 3-PS, 3-phosphoserine; OXPHOS, oxidative phosphorylation.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/582/eaav3048/DC1

    Fig. S1. Lack of effect of mTOR inhibition on α-SMA induction and the serine-glycine biosynthetic pathway, respectively.

    Fig. S2. Knockdown and knockout controls and evidence that TGF-β1 stimulation in fibroblasts is not associated with PERK activation.

    Fig. S3. ATF4 knockdown abrogates the TGF-β1–induced increase in glycine biosynthesis enzymes.

    Fig. S4. TGF-β1 stimulation increases glucose uptake and abundance of SLC2A1 transcripts, GLUT1, and glycolytic enzymes.

    Fig. S5. Exogenous serine does not rescue the inhibitory effects of ATP-competitive mTOR inhibition on TGF-β1–induced collagen deposition.

    Table S1. MetaCore pathways enriched in the rapamycin-insensitive mTOR module.

    Table S2. Primer sequences.

  • This PDF file includes:

    • Fig. S1. Lack of effect of mTOR inhibition on α-SMA induction and the serine-glycine biosynthetic pathway, respectively.
    • Fig. S2. Knockdown and knockout controls and evidence that TGF-β1 stimulation in fibroblasts is not associated with PERK activation.
    • Fig. S3. ATF4 knockdown abrogates the TGF-β1–induced increase in glycine biosynthesis enzymes.
    • Fig. S4. TGF-β1 stimulation increases glucose uptake and abundance of SLC2A1 transcripts, GLUT1, and glycolytic enzymes.
    • Fig. S5. Exogenous serine does not rescue the inhibitory effects of ATP-competitive mTOR inhibition on TGF-β1–induced collagen deposition.
    • Table S1. MetaCore pathways enriched in the rapamycin-insensitive mTOR module.
    • Table S2. Primer sequences.

    [Download PDF]

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