Research ArticleLung Disease

Mortality factor 4 like 1 protein mediates epithelial cell death in a mouse model of pneumonia

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Science Translational Medicine  28 Oct 2015:
Vol. 7, Issue 311, pp. 311ra171
DOI: 10.1126/scitranslmed.aac7793
  • Fig. 1. Morf4l1 accumulates in pneumonia.

    (A) Human lung tissues from patients with pneumonia (n = 8) and healthy patients (normal; n = 5) were analyzed for Morf4l1 protein expression by immunoblotting. β-Actin was used as a loading control. (B) Results of the densitometric analysis of bands from immunoblots. P= 0.029 versus normal tissue by the Mann-Whitney U test. N, normal; P, pneumonia. (C) Murine lung epithelial cells were exposed to various concentrations of LPS for 2 hours, and Morf4l1 was assayed by immunoblotting. (D) Steady-state levels of Morf4l1 mRNA determined by quantitative polymerase chain reaction (qPCR) after LPS exposure. (E) Murine lung epithelial cells were infected with P. aeruginosa strain 103 (PA103) at an MOI of 10 for 2 hours, and immunoreactivity of Morf4l1 or acetyltransferases was assayed by immunoblotting. (F) Steady-state levels of Morf4l1 mRNA determined by qPCR after 2 hours of infection with various MOI of PA103. Data in (B), (D), and (F) represent means ± SEM. Each panel in (B) to (F) represents at least n = 3 independent experiments.

  • Fig. 2. Morf4l1 is a labile protein degraded by the ubiquitin proteasome.

    (A) Murine lung epithelial cells were exposed to cycloheximide (CHX) alone or in combination with the proteasome inhibitor MG132 or the lysosomal inhibitor leupeptin for various times, and Morf4l1 was assayed by immunoblotting. (B) Results of the densitometric analysis of bands from immunoblots from (A). (C) Cells were transfected with wild-type (WT) V5-Morf4l1 or one of two V5-tagged Morf4l1 point Lys mutant plasmids (K136R, middle panels, or K143R, lower panels), and cells were exposed to CHX. Shown are the protein decay kinetics of WT and Morf4l1 mutant proteins. (D) Murine lung epithelial cells were transfected with WT V5-Morf4l1 (WT-V5) or a V5-tagged Morf4l1 point mutant plasmid (K143R mutant) (K143R-V5). Cell lysates were subjected to coimmunoprecipitation by immunoprecipitating (IP) proteins with V5 antibody followed by HA immunoblotting (IB). Shown is ubiquitination of Morf4l1-expressed proteins. Data in each panel represent n = 3 independent experiments. HA, hemagglutinin.

  • Fig. 3. SCFFbxl18 binds to Morf4l1 to mediate its polyubiquitination and degradation.

    (A) Murine lung epithelial cells were transfected with one of several V5-tagged plasmids encoding F-box proteins. The top panel shows the effects of various expressed plasmids on immunoreactive Morf4l1 levels in cells. The middle panel shows the relative cellular protein expression of plasmids by V5 immunoblotting, and the bottom panel shows β-actin. (B) Cells were transfected with various amounts of Fbxl18 or Fbxw18 plasmids. Shown are the results of the densitometric analysis of Morf4l1 bands from immunoblots after plasmid transfection. (C and D) Coimmunoprecipitation studies showing the interaction of Fbxl18 with complexes containing Morf4l1 and SCF E3 ligase components Skp1 and Cullin1. In the left panel, endogenous Fbxl18 was subjected to coimmunoprecipitation followed by immunoblotting with the indicated antibodies. In the right panel, endogenous Morf4l1 was subjected to immunoprecipitation followed by immunoblotting with the indicated antibodies. IgG, immunoglobulin G. (E) Cells were transfected with full-length WT V5-Morf4l1, a Morf4l1 N-terminal truncation plasmid (encoding residues 150 to 362), or a V5-tagged Morf4l1 double point mutant plasmid where leucine and glutamine in an IQ F-box motif were substituted with an alanine. After transfection, cell lysates were subjected to coimmunoprecipitation with Fbxl18 antibody followed by V5 immunoblotting. The top panel shows that, unlike WT Morf4l1, the expressed mutant proteins lack the ability to interact with the F-box protein. The middle and bottom panels show the relative expression of V5 proteins or Fbxl18 in cell lysates, respectively. WCL, whole cell lysate. (F) Cells were cotransfected with full-length WT V5-Morf4l1 or V5-tagged Morf4l1 point mutant plasmids with or without Fbxl18 plasmid. Shown in the top panel is the relative expression of Fbxl18 protein. The middle panel shows the levels of V5-tagged expressed mutant Morf4l1 proteins, and the bottom panel shows β-actin. (G) In vitro ubiquitination of Morf4l1. Shown are the effects of SCF-Fbxl18 on WT or K143R mutant Morf4l1 ubiquitination in vitro. Fbxw18 is a negative control. PolyUb, polyubiquitination. (H) Cells were transfected with Fbxl18 shRNA or control RNA and exposed to CHX. Shown graphically are the decay kinetics of Morf4l1 after Fbxl18 silencing. The data in each panel represent n = 3 independent experiments, except in panel (G) (n = 1).

  • Fig. 4. GCN5 acetylates and stabilizes Morf4l1.

    (A) Murine lung epithelial cells were exposed to anacardic acid (100 μM) to impair acetylation or to trichostatin A (1000 nM) to inhibit deacetylase activity for 4 hours with or without MG132; immunoreactivities of Morf4l1 and β-actin were then assayed. (B and C) Morf4l1 interaction with GCN5 by coimmunoprecipitation. (D) Morf4l1 protein decay is shown after expressing an empty vector or plasmids encoding Gcn5 or Gcn5-silencing plasmids and CHX treatment. Shown are immunoblots of Morf4l1, GCN5, and β-actin. (E) Cells were cotransfected with one of several Morf4l1 point mutant plasmids and Gcn5 plasmid. The levels of acetylated Morf4l1 are shown in the top panel, and cell lysates probed for V5 by immunoblotting are shown in the bottom panel. (F) Cells were transfected with WT V5-Morf4l1 or V5-Morf4l1 point mutant plasmid and then infected with P. aeruginosa (MOI, 10) to assess acetylated Morf4l1 (top row in each set) or total V5-Morf4l1 protein (bottom row in each set). (G) Cells were transfected with an empty vector or Gcn5 shRNA with P. aeruginosa infection (MOI, 10) and exposed to CHX. Shown graphically are the decay kinetics of Morf4l1 after Gcn5 silencing or bacterial infection. The data in each panel represent n = 3 independent experiments.

  • Fig. 5. Morf4l1 mediates P. aeruginosa–induced cell death.

    (A to C) Fluorescence-activated cell sorting analysis with 5-bromo-2′-deoxyuridine labeling of murine lung epithelial cells after expression of a pcDNA 3.1 vector (A) or pcDNA 3.1/Morf4l1 vector (B); the data were plotted in (C) to show cell cycle progression and apoptosis. (D and E) Cells were transfected with vector alone or various amounts of Morf4l1 plasmid and then processed for annexin V staining to assess apoptosis (D) or propidium iodine staining (E) to assess cell viability. (F) Cells were transfected with Morf4l1 deletion plasmids, and the number of viable cells was determined using trypan blue staining. (G) Cells were transfected with an empty vector, a plasmid encoding Morf4l1, or Morf4l1 shRNA and then infected with (or without) P. aeruginosa (MOI, 10). Shown are the numbers of viable cells after plasmid expression and infection. Data in (F) and (G) represent means ± SEM. *P < 0.05 versus 1–362 mutant (F). In (G), *P < 0.05 versus uninfected vector control or shRNA-infected group, and **P < 0.01 versus corresponding shRNA groups by the Mann-Whitney U test. (H) Immunoblot showing steady-state Morf4l1, Fbxl18, or β-actin protein in cells after transfection with Fbxl18 plasmid and then infection with P. aeruginosa or treatment. Data in each panel represent n = 3 independent experiments.

  • Fig. 6. Argatroban is a potent Morf4l1 inhibitor.

    (A) Morf4l1 crystal structure and potential docking site are shown. (B) Chemical structure of argatroban. (C)Morf4l1-argatroban docking; the small molecule embedded in Morf4l1 is shown in green. (D) A 2D diagram illustrates Morf4l1-argatroban interaction. Specifically, residues Gly220 and Thr277 within the Morf4-related gene domain (MRG) of Morf4l1 are essential for drug interactions. (E) ITC binding assays show argatroban binding to Morf4l1 with high affinity (66 nM). (F) Effect of argatroban on Morf4l1 activity (when presented within a protein complex) on acetyl–histone H4 in the cells. (G and H) Murine lung epithelial cells were transfected with Morf4l1 plasmid (G) or infected with P. aeruginosa (MOI, 10) (H) and then exposed to varying concentrations of argatroban, and effects on cell viability were analyzed. Data in each panel represent n = 3 independent experiments.

  • Fig. 7. Morf4l1 depletion or antagonism reduces cell death in experimental pneumonia.

    (A) Kaplan-Meier curves showing mouse survival after empty vector or Morf4l1 gene transfer in mice (n = 6 per group, *P = 0.0006 versus empty vector by the Mann-Whitney U test). (B and C) Lungs from mice in (A) were processed for TUNEL staining (B), which reveals the number of apoptotic cells (brown cellular staining in each group), and data were quantitated to show number of apoptotic cells in (C). The inset in (C) shows the relative protein expression of Morf4l1 in mouse lung tissue after gene transfer. *P < 0.05 versus empty vector by the Mann-Whitney U test. (D and E) Gene transfer was conducted in mice (n = 6 mice per group) with administration of an empty vector alone or an empty vector or Morf4l-silencing vector followed by P. aeruginosa infection (104 cfu per mouse, intratracheally). Shown in (D) are representative TUNEL stains of lung tissue and quantification of apoptotic cells in (E). A separate group of mice in (D) received argatroban (50 μg, intraperitoneally), and lungs were assessed by TUNEL staining. P < 0.05 versus empty vector with PA103 by the Kruskal-Wallis test. (F to H) Bronchoalveolar lavage from the mice in the above groups was processed for cell counts and protein concentration (F). *P < 0.05 versus empty vector with PA103. (G) The lungs were assayed for immunoreactivity of Morf4l1 and cleaved caspases. Mouse survival in each group was also determined, and Kaplan-Meier curves are shown in (H). Scale bars, 100 μm. *P = 0.002 versus empty vector.

  • Fig. 8. Pharmacological antagonism of Morf4l1 ameliorates LPS-mediated lung injury.

    (A) Lungs from mice were processed for TUNEL staining showing the numbers of apoptotic cells (brown cellular staining in each group). Mice were administered LPS (3 mg/kg, intratracheally), followed by phosphate-buffered saline (PBS) (left panels), dabigatran (5 μg per mouse) (middle panels), or argatroban (5 μg per mouse) (right panels). Cell death was monitored at a range of time points as indicated (n = 4 mice per group). (B)Data were quantitated to show the numbers of TUNEL-positive cells in (A). *P < 0.05 versus LPS alone. (C) Coagulation parameters prothrombin time (PT) and international normalized ratio (INR) were measured with clinical apparatus following the instructions of the manufacturer (ISTAT). (D and E) Dose effects of argatroban in LPS-mediated cell death in murine pneumonia. Each group of mice (n = 4 per group) was given LPS (5 mg/kg, intratracheally) followed by different amounts of argatroban. Lung tissues were collected at 6 hours for TUNEL staining. Shown in (D) are representative TUNEL stains of lung tissue and quantification of apoptotic cells in (E). ** P < 0.05 versus 0 dose argatroban by the Kruskal-Wallis test. (F) Kaplan-Meier curves for mice exposed to LPS and treated with dabigatran or argatroban (n = 8 animals per group; the experiment was terminated at 24 hours). *P < 0.0001 versus LPS alone by the Kruskal-Wallis test. (G) The lungs from (A) were assayed for immunoreactivity of a selected panel of apoptosis and stress proteins, and the inset shows the results of the assay. Densitometry results of each protein were plotted. Scale bars, 100 μm. *P < 0.05 and **P < 0.01 versus PBS by the Kruskal-Wallis test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/311/311ra171/DC1

    Materials and Methods

    Fig. S1. Endotoxin and P. aeruginosa increase Morf4l1 protein levels posttranslationally in lung cells.

    Fig. S2. Bacterial pathogens trigger Morf4l1 acetylation.

    Fig. S3. Effect of Morf4l1 depletion or antagonism on bacterial loads.

    Fig. S4. Effect of Morf4l1 depletion or antagonism on the Fas cell death pathway.

    Fig. S5. Morf4l1 mediates cell death in pneumonia.

  • Supplementary Material for:

    Mortality factor 4 like 1 protein mediates epithelial cell death in a mouse model of pneumonia

    Chunbin Zou,* Jin Li, Sheng Xiong, Yan Chen, Qin Wu, Xiuying Li, Nathaniel M. Weathington, SeungHye Han, Courtney Snavely, Bill B. Chen, Rama K. Mallampalli*

    *Corresponding author. E-mail: mallampallirk{at}upmc.edu (R.K.M.); zouc{at}upmc.edu (C.Z.)

    Published 28 October 2015, Sci. Transl. Med. 7, 311ra171 (2015)
    DOI: 10.1126/scitranslmed.aac7793

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Endotoxin and P. aeruginosa increase Morf4l1 protein levels posttranslationally in lung cells.
    • Fig. S2. Bacterial pathogens trigger Morf4l1 acetylation.
    • Fig. S3. Effect of Morf4l1 depletion or antagonism on bacterial loads.
    • Fig. S4. Effect of Morf4l1 depletion or antagonism on the Fas cell death pathway.
    • Fig. S5. Morf4l1 mediates cell death in pneumonia.

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