Research ArticleLung Disease

The proinflammatory role of HECTD2 in innate immunity and experimental lung injury

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Science Translational Medicine  08 Jul 2015:
Vol. 7, Issue 295, pp. 295ra109
DOI: 10.1126/scitranslmed.aab3881
  • Fig. 1. HECTD2 targets PIAS1 for polyubiquitination, thereby increasing NF-κB signaling.

    (A) Endogenous PIAS1 protein half-life study with MG132 or leupeptin (n = 3). CHX, cycloheximide. (B) Immunoblotting showing levels of endogenous PIAS proteins and V5-HECTD2 after ectopic HECTD2 plasmid expression (n = 3). (C) Endogenous PIAS1 protein half-life determination with empty plasmid or HECTD2 expression (top panel); endogenous PIAS1 protein half-life determination with control (CON) shRNA or HECTD2 shRNA expression (bottom panel) (n = 3). (D) In vitro ubiquitination assay. Purified E1 and E2 components were incubated with V5-PIAS1, HECTD2, and the full complement of ubiquitination reaction components (second lane) showing polyubiquitinated PIAS1 proteins (n = 2). (E to G) NF-κB promoter activity assays. 293T cells were cotransfected with Cignal NF-κB dual luciferase reporter plasmids along with empty or HECTD2 plasmid or control shRNA, HECTD2 shRNA, or PIAS1 shRNA 24 hours before HECTD2 plasmid expression. Twenty-four hours later, cells were treated with LPS (10 μg/ml), TNF (10 ng/ml), or IFN-γ (10 ng/ml) for an additional 6 hours (F and G) or 18 hours. Cells were then collected and assayed for luciferase activity to evaluate NF-κB promoter activity. Means ± SEM (n = 3). (H) MLE cells were treated with LPS in a time- or dose-dependent manner; cells were then collected and assayed for HECTD2, PIAS, and actin immunoblotting. Endogenous HECTD2 was also immunoprecipitated (IP) and followed by PIAS1 and PIAS4 immunoblotting (n = 2). Source data in fig. S10 and table S2.

  • Fig. 2. PIAS1 phosphorylation is required for HECTD2 targeting.

    (A) Endogenous PIAS1 was immunoprecipitated and followed by GSK3β and phosphoserine antibody immunoblotting (IB) (n = 2). IgG, immunoglobulin G. (B and C) MLE cells were treated with LPS in a time- or dose-dependent manner; cells were then collected and assayed for HECTD2, PIAS, and actin immunoblotting. Endogenous PIAS1 was also immunoprecipitated and followed by GSK3β, phosphoserine, and phosphothreonine immunoblotting (n = 2). (D) Endogenous PIAS1 protein half-life determination with control shRNA or GSK3β shRNA overexpression (n = 3). (E) MLE cells were transfected with increasing amounts of WT or constitutively activated GSK3β hypermutant plasmids for 18 hours before PIAS1 immunoblotting. The arrow indicates the overexpressed GSK3β (n = 2). (F) Endogenous PIAS1 protein half-life determination with WT GSK3β or hyperactive GSK3β plasmid overexpression. Arrow indicates the overexpressed GSK3β (n = 2). (G) Immunoblotting showing levels of endogenous GSK3β and PIAS1 protein in MLE cells transfected with either control shRNA or GSK3β shRNA followed by LPS treatment. Endogenous PIAS1 was also immunoprecipitated and followed by phosphoserine and phosphothreonine immunoblotting. (H) In vitro GSK3β kinase assay using PIAS1 as a substrate. GSK3β*, heat-inactivated GSK3β (n = 2). (I) Protein half-life of WT, S13A, and S17A PIAS1 (n = 3). (J) MLE cells were transfected with WT, S13A, or S17A PIAS1 before being treated with LPS for up to 6 hours. Cells were then collected and assayed for V5-PIAS1. Overexpressed V5-PIAS1 was also immunoprecipitated using V5 antibody and followed by phosphoserine immunoblotting (n = 2). (K) Four biotin-labeled peptides were prebound to streptavidin and served as the bait for HECTD2 or GSK3β binding. After washing, proteins were eluted and processed for V5-HECTD2 or GSK3β immunoblotting (n = 2). Source data in fig. S11.

  • Fig. 3. HECTD2 contains a naturally occurring polymorphism at A19, which mislocalizes to the cytosol.

    (A) In vitro ubiquitination assay using V5-PIAS1 as the substrate and HECTD2WT and HECTD2A19P as the E3 ligase (n = 3). (B) Endogenous PIAS1 protein was immunoprecipitated from cell lysate using PIAS1 antibody and coupled to protein A/G beads. PIAS1 beads were then incubated with in vitro synthesized products expressing his-V5-HECTD2 mutants (top). After washing, proteins were eluted and processed for V5-HECTD2 immunoblotting (bottom) (n = 2). (C) MLE cells were transfected with increasing amounts of WT or A19P HECTD2 plasmids for 18 hours before being assayed for PIAS1 protein immunoblotting (n = 3). (D and E) MLE cells were transfected with an inducible HECTD2WT or HECTD2A19P plasmid under control of exogenous doxycycline. Cells were treated with doxycycline for various times and dose. Cells were then collected, and cell lysates were analyzed for HECTD2 and PIAS1 by immunoblotting (n = 2). (F) Half-life study of endogenous PIAS1 upon HECTD2WT or HECTD2A19P overexpression (n = 2). (G) 293T cells were cotransfected with Cignal NF-κB reporter plasmids and empty, HECTD2WT, or HECTD2A19P plasmid for 18 hours. Cells were then exposed to LPS or TNF for additional 18 hours before being assayed for NF-κB promoter activity. Means ± SEM (n = 3). (H) MLE cells were cotransfected with CFP-PIAS1 and YFP-HECTD2WT or YFP-HECTD2A19P. YFP-HECTD2WT and YFP-HECTD2A19P nuclear/cytosol fluorescent signals were measured and quantified (n > 20, right graph, *P < 0.0001 compared to WT). Cells were also subjected to irreversible photobleaching of YFP acceptor signal using a 514-nm laser. The emission fluorescence levels of both the donor CFP-PIAS1 and acceptor YFP-HECTD2WT or YFP-HECTD2A19P before and after acceptor photobleaching are shown in the lower panel; the region of interest across the nucleus is marked with a red arrow. The green arrow on the graph indicates donor CFP-PIAS1 signal before and after the photobleaching. Scale bar, 10 μm (n > 10). Source data in fig. S12 and table S3.

  • Fig. 4. HECTD2A19P is a loss-of-function E3 ligase polymorphism in vivo.

    C57BL/6J mice were administered intratracheally with Lenti-control, Lenti- HECTD2WT, or Lenti-HECTD2A19P [107 plaque-forming units (PFU) per mouse] for 144 hours, and mice were inoculated intratracheally with PA103 (104 PFU per mouse) for 18 hours. Mice were then euthanized, and lungs were lavaged with saline, harvested, and then homogenized. (A to C) Lavage protein, cell count, and bacteria count were measured. BAL, bronchoalveolar lavage. (D to F) Lavage cytokine secretion was measured. IL-6, interleukin-6. (G) Hematoxylin and eosin (H&E) staining was performed on lung samples from (A). Scale bar, 100 μm; original magnification, ×10. (H) PIAS1, HECTD2, and actin immunoblots from homogenized lung samples. Data are average of two experiments (Student’s t test on means ± SEM). (A to G) n = 5 to 12 mice per group. Source data in fig. S13 and table S4.

  • Fig. 5. PIAS1 knockdown induces lung injury in vivo.

    C57BL/6J mice were infected intratracheally with Lenti-control shRNA or Lenti-PIAS1 shRNA (107 PFU per mouse) for 144 hours, and mice were inoculated with LPS (intratracheally, 3 mg/kg) for 18 hours. Mice were euthanized, and lungs were lavaged with saline, harvested, and then homogenized. (A to C) Lavage protein, cell count, and lung protein immunoblots were measured. (D to F) Lavage cytokine secretion was measured. (G) H&E staining was performed on lung samples from (A). Scale bar, 100 μm; original magnification, ×10 (Student’s t test on means ± SEM). (A to G) n = 5 to 7 mice per group. Source data in fig. S13 and table S5.

  • Fig. 6. HECTD2 knockdown ameliorates Pseudomonas-induced lung injury in vivo.

    C57BL/6J mice were infected intratracheally with Lenti-control shRNA or Lenti-HECTD2 shRNA (107 PFU per mouse) for 144 hours, and mice were inoculated with PA103 [104 colony-forming units (CFU) per mouse] for 18 hours. Mice were euthanized, and lungs were lavaged with saline, harvested, and then homogenized. (A to C) Lavage protein, cell count, and bacteria count were measured. (D to F) Lavage cytokine secretion was measured. (G) Survival studies of mice that were administered PA103 (intratracheally, 105 PFU per mouse, n = 8 mice per group) were determined (time, hours). Mice were carefully monitored over time; moribund, preterminal animals were immediately euthanized and recorded as deceased. Kaplan-Meier survival curves were generated using SPSS software (P = 0.001). (H) H&E staining was performed on lung samples from (A). (I) PIAS1, HECTD2, and actin immunoblots from homogenized lung samples. Scale bar, 100 μm; original magnification, ×10. Data are average of two experiments (Student’s t test on means ± SEM). (A to G) n = 6 to 9 mice per group. NS, not significant. Source data in fig. S13 and table S6.

  • Fig. 7. Anti-inflammatory activity of a HECTD2 small-molecule inhibitor.

    (A) Structural analysis of the HECTD2-HECT domain revealed a major cavity within the C terminus of the HECT domain. (B) Docking study of candidate inhibitor BC-1382 with the HECTD2-HECT domain. (C) HECTD2 protein was immunoprecipitated using HECTD2 antibody and captured with protein A/G beads from MLE lysates. HECTD2 beads were extensively washed before exposure to BC-1382 at different concentrations (10−4 to 10 μM). Purified PIAS1 protein was then incubated with drug bound to HECTD2 beads overnight. Beads were washed, and proteins were eluted and resolved on SDS–polyacrylamide gel electrophoresis. The relative amounts of PIAS1 detected in the pull-downs were normalized to loading and quantified (n = 2). (D to H) C57BL/6J mice were infected intratracheally with PA103 (104 PFU per mouse). BC-1382 was given through intraperitoneal injection (10 mg/kg) at the same time. Eighteen hours later, mice were sacrificed, and lungs were lavaged with saline, harvested, and then homogenized. Lavage bacterial count, protein, cell count, and cytokine secretion were measured in (D) to (G). (H) H&E staining was performed on lung samples (scale bar, 100 μm; original magnification, ×10). (I) PIAS1, HECTD2, and actin immunoblots from homogenized lung samples. Data are average of two experiments (Student’s t test on means ± SEM). (D to H) n = 5 to 10 mice per group. Source data in fig. S13 and table S7.

  • Fig. 8. One mechanism by which microbial infection or stimuli can robustly trigger inflammation is to decrease anti-inflammatory proteins such as PIAS1 in cells.

    Specifically, during microbial infection, HECTD2 (R397) targets PIAS1 for its ubiquitination at K30; this process is facilitated by GSK3β phosphorylation of PIAS1 at S17. In this pathway, WT HECTD2 potently activates cytokine-driven inflammation, whereas we identified a naturally occurring, mislocalized, hypofunctional variant of HECTD2 (A19P) that lowers the amount of cytokine secretion. A small-molecule HECTD2 inhibitor, BC-1382, decreased inflammation in an animal model of ARDS by antagonizing the actions of HECTD2 on PIAS1, allowing PIAS1 to continue to suppress cytokine signaling.

  • Table 1. rs7081569 SNP.

    SNP analysis of HECTD2 protein indicating an A19P polymorphism (rs7081569) (noncoding strand is shown at http://www.ncbi.nlm.nih.gov/projects/SNP).

    Chromosome
    10 position
    91410493 (+)
    SequenceGTGGCGGCGG/CCCGCGCCTGA
    AA changeMSEAVRVPSPATPLVVAAA/PAPEERKGKESEREKLPPIVS
  • Table 2. Demographic characteristics by rs7081569 polymorphism.

    N/A, not applicable.

    ARDS
    (n = 63)
    P1000 Genomes Project*
    (n = 1092)
    P
    CC
    (n = 0)
    CG
    (n = 0)
    GG
    (n = 63)
    CC
    (n = 6)
    CG
    (n = 174)
    GG
    (n = 912)
    Male sex (%)47N/A1750480.267
    Race (%)
      European95332836
      Hispanic001517
      Asian0172427
      African5N/A5033200.012

    *1000 Genomes Project, www.1000genomes.org/.

    P value by χ2 test.

    • Table 3. Association of rs7081569 polymorphism in ARDS cohort (Europeans, n = 60) and control Europeans (n = 439).

      1000 Genomes Project, www.1000genomes.org/. P value by χ2 test.

      SNP genotypeALI (%)Healthy controls (%)P
      GG60 (100)328 (87)
      CG/CC0 (0)51 (13)0.003

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/7/295/295ra109/DC1

      Fig. S1. PIAS1 degradation occurs in a ubiquitin-dependent manner and through the proteasome.

      Fig. S2. K30 is the ubiquitin acceptor site within PIAS1.

      Fig. S3. Deletional mapping study identifying PIAS1 binding site within HECTD2.

      Fig. S4. R397 is the preferred binding site for PIAS1.

      Fig. S5. HECTD2 interacts with the first 20 amino acids of PIAS1.

      Fig. S6. HECTD2A19P and deletional mutant localization study.

      Fig. S7. FRET study of HECTD2 chimera proteins with PIAS1.

      Fig. S8. BC1382 exhibits anti-inflammatory activity through preservation of PIAS1.

      Fig. S9. BC1382 ameliorates LPS-induced lung injury.

      Fig. S10. Source data for Fig. 1 (A to D and H).

      Fig. S11. Source data for Fig. 2 (A to K).

      Fig. S12. Source data for Fig. 3 (A to F).

      Fig. S13. Source data for Figs. 4H, 5C, 6I, and 7 (C and I).

      Table S1. Source sequencing data for Table 3.

      Table S2. Source data for Fig. 1 (E to G).

      Table S3. Source data for Fig. 3G.

      Table S4. Source data for Fig. 4 (A to F).

      Table S5. Source data for Fig. 5 (A, B, and D to F).

      Table S6. Source data for Fig. 6 (A to G).

      Table S7. Source data for Fig. 7 (C to G).

    • Supplementary Material for:

      The proinflammatory role of HECTD2 in innate immunity and experimental lung injury

      Tiffany A. Coon, Alison C. McKelvey, Travis Lear, Shristi Rajbhandari, Sarah R. Dunn, William Connelly, Joe Y. Zhao, SeungHye Han, Yuan Liu, Nathaniel M. Weathington, Bryan J. McVerry, Yingze Zhang, Bill B. Chen*

      *Corresponding author. E-mail: chenb{at}upmc.edu

      Published 8 July 2015, Sci. Transl. Med. 7, 295ra109 (2015)
      DOI: 10.1126/scitranslmed.aab3881

      This PDF file includes:

      • Fig. S1. PIAS1 degradation occurs in a ubiquitin-dependent manner and through the proteasome.
      • Fig. S2. K30 is the ubiquitin acceptor site within PIAS1.
      • Fig. S3. Deletional mapping study identifying PIAS1 binding site within HECTD2.
      • Fig. S4. R397 is the preferred binding site for PIAS1.
      • Fig. S5. HECTD2 interacts with the first 20 amino acids of PIAS1.
      • Fig. S6. HECTD2A19P and deletional mutant localization study.
      • Fig. S7. FRET study of HECTD2 chimera proteins with PIAS1.
      • Fig. S8. BC1382 exhibits anti-inflammatory activity through preservation of PIAS1.
      • Fig. S9. BC1382 ameliorates LPS-induced lung injury.
      • Fig. S10. Source data for Fig. 1 (A to D and H).
      • Fig. S11. Source data for Fig. 2 (A to K).
      • Fig. S12. Source data for Fig. 3 (A to F).
      • Fig. S13. Source data for Figs. 4H, 5C, 6I, and 7 (C and I).
      • Table S1. Source sequencing data for Table 3.
      • Table S2. Source data for Fig. 1 (E to G).
      • Table S3. Source data for Fig. 3G.
      • Table S4. Source data for Fig. 4 (A to F).
      • Table S5. Source data for Fig. 5 (A, B, and D to F).
      • Table S6. Source data for Fig. 6 (A to G).
      • Table S7. Source data for Fig. 7 (C to G).

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