Research ArticlePharmacology

Identification of a Lysosomal Pathway That Modulates Glucocorticoid Signaling and the Inflammatory Response

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Science Signaling  05 Jul 2011:
Vol. 4, Issue 180, pp. ra44
DOI: 10.1126/scisignal.2001450
  • Fig. 1

    The anti-inflammatory effect of CQ was partially mediated by GR. (A) In THP-1 cells exposed to LPS (1 μg/ml), CQ (50 μM) reduced the mRNA abundance of IL-1β and IL-6 (measured by real-time PCR) compared to PBS (vehicle)–treated cells exposed to LPS (1 μg/ml). Data represent the means and SD (n ≥ 3 samples). (B) Dex (100 nM) repressed IL-1β and IL-6 mRNA transcription in LPS-stimulated THP-1 cells. Data represent the means and SD (n ≥ 3 samples). (C) Dose effect of CQ in activation of the MMTV-luciferase reporter in AD293 cells without added exogenous glucocorticoid. Cells were treated with CQ for 16 hours. RLU, relative luciferase units that were normalized with Renilla luciferase. Data represent the means and SD (n ≥ 3 samples). Statistical significance between the 50 μM and 0 μM CQ samples is indicated. (D) Dose effect of CQ in repression of the AP-1 luciferase reporter in AD293 cells exposed to PMA (6.25 ng/ml) in the absence of exogenous glucocorticoid. Data represent the means and SD (n ≥ 3 samples). Statistical significance between the 50 μM and 0 μM CQ samples is indicated. (E) Knockdown of GR by siRNA decreased the repression by CQ of IL-1β and IL-6 mRNA in LPS-stimulated THP-1 cells. Data represent the means and SD (n ≥ 3 samples). NC siRNA, negative control siRNA. (F) GR knockdown efficiency was measured at the mRNA level by real-time PCR and at the protein level by Western blotting with the PA1-511A antibody that recognizes human GR. β-Actin was used as a loading control. mRNA data represent the means and SD of n ≥ 3 samples and the Western blotting data are representative of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 2

    CQ enhances glucocorticoid-mediated GR signaling. (A) CQ and Dex competition assays show that CQ did not bind to GR. Data are representative of three experiments. (B) CQ (50 μM) enhanced Dex-stimulated transactivation of the MMTV reporter (MMTV-Luc) or transrepression of the AP-1 reporter (AP-1–Luc) in AD293 cells. For the AP-1 reporter assay, the cells were stimulated with PMA (6.25 ng/ml). Data represent the means and SD (n ≥ 3 samples). (C) Removal of endogenous GR ligands from serum with charcoal (stripped medium) decreased the effectiveness of CQ (50 μM) on enhancing GR-mediated changes in gene expression in AD293 cells. *P < 0.05 compared to CQ treatment in the presence of normal medium. Data represent the means and SD (n ≥ 3 samples). (D) CQ (50 μM) enhanced glucocorticoid (Dex, 100 nM)–mediated repression of IL-1β and IL-6 mRNA in THP-1 cells exposed to LPS (1 μg/ml). *P < 0.05 compared to Dex-alone treatment. Data represent the means and SD (n ≥ 3 samples). (E) Mean clinical arthritis scores of mice treated with vehicle (PBS, n = 10), Dex (4 μg per mouse, n = 13), CQ (400 μg per mouse, n = 10), or Dex + CQ [(4 μg + 400 μg) per mouse, n = 15]. *P < 0.05, **P < 0.01. (F) Representative pictures of the feet of arthritic mice before and after CQ + Dex treatment.

  • Fig. 3

    Gene expression profiling of THP-1 cells exposed to CQ, Dex, or both. (A) Top 70 GR-regulated genes that showed a synergistic effect upon treatment with CQ (50 μM) plus Dex (100 nM) in THP-1 cells stimulated with LPS (1 μg/ml). Changes in gene expression of the genes listed in bold capital letters were validated with real-time PCR. (B) Percentage of Dex-induced and Dex-repressed genes that have enhanced or synergistic effects with CQ. Enhanced responses were those that in the presence of both Dex and CQ exhibited a greater response than either drug alone (Dex + CQ cotreatment > Dex alone or CQ alone). Synergistic responses represented those in which in the presence of both Dex and CQ, the response more than exceeded the sum of the response to either drug individually (Dex + CQ cotreatment > Dex alone + CQ alone). (C) Validation of the synergistic effect on the indicated repressed or activated genes by real-time PCR. Selected genes represent a spectrum of Dex-regulated genes (from low to high); for other validations, see fig. S4. Genes include those particularly relevant for inflammatory responses and represent a range of GR-responsive genes.

  • Fig. 4

    The V-ATPase inhibitor bafilomycin A1 potentiated glucocorticoid signaling by inhibiting lysosomal function. (A) CQ interferes with lysosomal function by increasing lysosomal internal pH. AD293 cells were treated with the indicated concentrations of CQ for 4 hours and then stained with LysoTracker Red, which labels all low-pH compartments. The rectangles represent 1.5× magnifications of regions from within the images; images were obtained by 100×/oil objective lens. Scale bars, 10 μm. (B) Bafilomycin A1 (Baf A1, 100 nM) and concanamycin A (CocA, 1 nM) potentiated glucocorticoid (Dex, 10 nM)–stimulated transactivation of the MMTV reporter in AD293 cells. **P < 0.01 compared to Dex induction in the presence of dimethyl sulfoxide (DMSO). Data represent the means and SD (n ≥ 3 samples). (C) Baf A1 (100 nM) synergized with glucocorticoid (Dex, 100 nM) in repression of IL-1β and IL-6 mRNA transcription in THP-1 cells in the presence of LPS (1 μg/ml). **P < 0.01 compared to Dex induction treatment. Data represent the means and SD (n ≥ 3 samples). (D) Knockdown of V-ATPase components by siRNA potentiated glucocorticoid (Dex, 10 nM)–mediated transactivation of the MMTV reporter in AD293 cells. Dashed line indicates the transactivation level of negative control siRNA (NC siRNA). *P < 0.05, **P < 0.01, compared to NC siRNA in cells treated with Dex; average siRNA knockdown efficiency was 70 ± 10% as measured by real-time PCR (fig. S6). Data represent the means and SD (n ≥ 3 samples).

  • Fig. 5

    Lysosomal biogenesis master regulator TFEB regulates GR activity. (A) Knockdown of TFEB by siRNA potentiated glucocorticoid (Dex, 10 nM)–mediated transactivation of the MMTV reporter in AD293 cells. **P < 0.01, compared to Dex-treated cells with control siRNA. Data represent the means and SD of n ≥ 3 samples. (B) Knockdown of TFEB by siRNA decreased proinflammatory cytokines IL-6 and tumor necrosis factor–α (TNFα) mRNA abundances, with or without Dex (100 nM), in THP-1 cells exposed to LPS (1 μg/ml). TFEB knockdown efficiency was measured at the mRNA level. *P < 0.05, **P < 0.01, compared to control siRNA. Data represent the means and SD (n ≥ 3 samples). (C) Overexpression of TFEB in AD293 cells inhibited glucocorticoid (Dex, 10 nM)–mediated induction of the MMTV reporter. The TFEB-expressing plasmid (0 to 20 ng) balanced with an equal amount of vehicle plasmid was used in transfection. *P < 0.05, **P < 0.01, compared to Dex-treated vehicle control samples (TFEB 0 ng). Data represent the means and SD (n ≥ 3 samples). (D) Cotransfection of TFEB (100 to 1000 ng of 3×FLAG-TFEB per well, six-well plate) with GR (500 ng per well, six-well plate) gradually decreased the amount of GR, with or without GR ligand (Dex, 100 nM) in AD293 cells. GR was detected with the PA1-511A antibody. Data shown are representative of three experiments. (E) Knockdown of TFEB by siRNA increased the abundance of GR in U2OS cells stably expressing GR. Data shown are representative of two experiments. GR mRNA was not affected by TFEB siRNA as measured by real-time PCR. Data from two experiments are shown. (F) CQ (50 μM) increased the amount of GR in U2OS cells stably expressing GR, with or without glucocorticoid (Dex, 100 nM). β-Actin served as the loading control. Data shown are representative of three experiments.

  • Fig. 6

    Lysosomes regulate AR and ER activity. (A) CQ enhanced the transactivation activity of the AR ligand R1881 (10 nM) on the MMTV reporter in AD293 cells. **P < 0.01 compared to vehicle control R1881–treated cells. Data represent the means and SD (n ≥ 3 samples). (B) CQ enhanced the transactivation activity of the ER ligand E2 (10 nM) on the ERE reporter in AD293 cells. **P < 0.01 compared to vehicle control E2–treated cells. Data represent the means and SD (n ≥ 3 samples). (C) Overexpression of TFEB decreased the AR (R1881, 10 nM)–mediated activation of the MMTV reporter in AD293 cells. *P < 0.05, **P < 0.01 compared to vehicle control (TFEB 0 ng) R1881–treated cells. Data represent the means and SD (n ≥ 3 samples). (D) Overexpression of TFEB decreased the ER (E2, 10 nM)–mediated activation of the MMTV reporter in AD293 cells. *P < 0.05 compared to vehicle control E2–treated cells. Data represent the means and SD (n ≥ 3 samples). (E) Cotransfection of TFEB (250 ng of 3×FLAG-TFEB per well in 24-well plate) with AR (100 ng/well, 24-well plate) decreased the abundance of AR, with or without the AR ligand R1881 (10 nM), in AD293 cells. AR was detected with the 441 antibody; endogenous p53 and β-catenin were detected with the DO-1 and 610154 antibodies, respectively. (F) Cotransfection of TFEB (250 ng of 3×FLAG-TFEB in 24-well plate) with ER (100 ng/well, 24-well plate) decreased the abundance of ER, with or without the ER ligand E2 (10 nM) in AD293 cells. ER was detected with the F10 antibody.

  • Fig. 7

    A lysosomal pathway contributes to degradation of cytoplasmic GR. (A) Pulse-chase assay of unliganded GR in AD293 cells. AD293 cells were transfected with 200 ng of Halo-GR per well in 24 wells. Cells were pulse-labeled with 20 nM TMR ligand and then chased with 10 μM succinimidyl ester (O4) ligand. Upper panels, fluorescent TMR-Halo-GR SDS-PAGE scanning images. Lower panels, Western blots. GR was detected by PA1-511A antibody. CQ (50 μM) and MG132 (10 μM) were added at the same time that chase began. Data shown are representative of three experiments. (B) Pulse-chase assay of liganded GR in AD293 cells. Cells were transfected, labeled, and chased as described for (A). Upper panel, TMR-Halo-GR bands; lower panel, Western blots. Dex (100 nM), CQ (50 μM), and MG132 (10 μM) were added at the same time that chase began. Data shown are representative of three experiments. (C) Live-cell images of Halo-GR in the pulse-chase assay. AD293 cells were passaged into 40-mm glass-bottomed dishes and transfected with 0.5 μg of Halo-GR. Halo-GR was labeled with 2 nM TMR ligand (red fluorescence) and chased with 10 μM O4 ligand. Images were obtained with a 40× confocal microscope. Scale bar, 10 μm. See videos S1 and S2. (D) Pulse-chase assay of GR AD293 cells in which TFEB was knocked down. AD293 cells were transfected with 200 ng of Halo-GR together with 20 nM target siRNA in 24-well plates. Cells were labeled and chased as described in (A). Upper panel, TMR-Halo-GR bands; lower panel, Western blots. Data shown are representative of two experiments. (E) Live-cell images of Halo-GR and LAMP1-YFP in AD293 cells. Cells were transfected with 500 ng of Halo-GR and 500 ng of LAMP1-YFP in 40-mm glass-bottomed dishes. Cells were pulse-labeled by 20 nM TMR ligand; images were obtained with a 40× confocal microscope. Scale bar, 5 μm. Arrows indicate lysosomal location. (F) Dynamic association of Halo-GR and lysosomes in AD293 cells. Cells were transfected and labeled as in (E). Images were obtained with a 60× confocal microscope. Scale bar, 5 μm. Arrows point to the yellow sparks of lysosomes, indicating GR movement. See video S3.

Additional Files

  • Supplementary Materials for:

    Identification of a Lysosomal Pathway That Modulates Glucocorticoid Signaling and the Inflammatory Response

    Yuanzheng He,* Yong Xu, Chenghai Zhang, Xiang Gao, Karl J. Dykema, Katie R. Martin, Jiyuan Ke, Eric A. Hudson, Sok Kean Khoo, James H. Resau, Arthur S. Alberts, Jeffrey P. MacKeigan, Kyle A. Furge, H. Eric Xu*

    *To whom correspondence should be addressed. E-mail: eric.xu{at}vai.org (H.E.X.); ajian.he{at}vai.org (Y.H.)

    This PDF file includes:

    • Fig. S1. Activity of CQ and amodiaquine (AQ).
    • Fig. S2. Effect of CQ and bafilomycin A1 on GR localization in AD293 cells.
    • Fig. S3. CQ enhances glucocorticoid-mediated GR signaling.
    • Fig. S4. Microarray analysis of the effect of CQ/Dex on gene expression in THP-1 cells.
    • Fig. S5. CQ inhibition of lysosomes leads to accumulation of autophagosomes and autolysosomes.
    • Fig. S6. Knockdown efficiency of the siRNAs targeting components of the VATPase.
    • Fig. S7. CQ stabilizes GR in AD293 cells.
    • Fig. S8. The effect of CQ on a β-catenin�activated reporter gene.
    • Fig. S9. Effect of the proteasome inhibitor MG132 and the lysosomal inhibitor CQ on GR activation in AD293 cells and COS7 cells.
    • Table S1. Top 70 genes regulated by Dex in LPS-stimulated THP-1 cells.
    • Table S2. Top 70 genes regulated by CQ in LPS-stimulated THP-1 cells.
    • Table S3. Top 70 genes regulated by Dex + CQ in LPS-stimulated THP-1 cells.
    • Table S4. Top 70 genes showing a synergistic transactivation effect upon Dex + CQ treatment in LPS-stimulated THP-1 cells.
    • Table S5. Top 70 genes showing a synergistic transrepression effect upon Dex + CQ treatment in LPS-stimulated THP-1 cells.
    • Table S6. Real-time PCR primers for target genes.
    • Table S7. siRNA target sequences.
    • Descriptions for Videos S1 to S3

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    Other Supplementary Material for this manuscript includes the following:

    • Video S1 (.avi format). Pulse chase of Halo-GR in AD293 cells with PBS vehicle.
    • Video S2 (.avi format). Pulse chase of Halo-GR in AD293 cells exposed to CQ.
    • Video S3 (.avi format). Dynamic association of GR and lysosomes in AD293 cells.

    [Download Videos S1 to S3 (Compressed)]

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    63.8 MB (Video S1 decompressed); 63.8 MB (Video S2 decompressed); 122.6 MB (Video S3 decompressed)


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    Citation: Y. He, Y. Xu, C. Zhang, X. Gao, K. J. Dykema, K. R. Martin, J. Ke, E. A. Hudson, S. K. Khoo, J. H. Resau, A. S. Alberts, J. P. MacKeigan, K. A. Furge, H. E. Xu, Identification of a Lysosomal Pathway That Modulates Glucocorticoid Signaling and the Inflammatory Response. Sci. Signal. 4, ra44 (2011).

    © 2011 American Association for the Advancement of Science

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