A novel pathway of LPS uptake through syndecan-1 leading to pyroptotic cell death

  1. Shigetoshi Yokoyama
  2. Yan Cai
  3. Miyuki Murata
  4. Takeshi Tomita
  5. Mitsuhiro Yoneda
  6. Lei Xu
  7. Aprile L Pilon
  8. Raul E Cachau
  9. Shioko Kimura  Is a corresponding author
  1. National Cancer Institute, National Institutes of Health, United States
  2. APCBIo Innovations Inc., United States
  3. Frederick National Laboratory for Cancer Research, Leidos Biomedical Inc., United States
6 figures, 1 table and 4 additional files

Figures

SCGB3A2-induced suppression of LLC cell proliferation.

(A) Effect of SCGB3A2 on proliferation of LLC cells. Cells were maintained without serum for 24 hr, followed by 1% FBS-RPMI1640 media with or without mouse SCGB3A2 (1 μg/ml). CCK8 assay was carried …

https://doi.org/10.7554/eLife.37854.003
Figure 2 with 1 supplement
SCGB3A2 as an LPS binding protein.

(A) CCK8 analysis using various concentrations as shown in the bottom (pg, ng/ml) of smooth LPS (E.coli O111:B4 serotype) and rough LPS (Ra-LPS) after 72 hr in culture. C; control without any …

https://doi.org/10.7554/eLife.37854.004
Figure 2—figure supplement 1
Analysis of LPS-SCGB3A2 complex.

(A) CCK8 analysis using various recombinant SCGB3A2s (1 µg/ml) obtained from different sources/batches. LLC cells grown in 1% FBS-RPMI 1640 medium were harvested at 72 hr and analyzed. Averages ± SD …

https://doi.org/10.7554/eLife.37854.005
Figure 3 with 1 supplement
SCGB3A2 binding to LLC cells through HS of SDC1.

(A) Schematic model of a human protein array for the isolation process of candidate genes shown as a Venn diagram. (B) Co-immunoprecipitation assay of SCGB3A2-FLAG and SDC1-Myc-His in COS-1 cells. …

https://doi.org/10.7554/eLife.37854.006
Figure 3—source data 1

Results of whole protein-protein interaction array.

https://doi.org/10.7554/eLife.37854.008
Figure 3—figure supplement 1
Cell surface expression of SDC1 and validation of sh-SDC1 and ARH-77-mSDC1 clones.

(A) Representative IHC for SDC1 in lung metastasized LLC cells in mice. Bar = 200 µm (upper), 50 µm (bottom). Bottom is magnified image of red rectangle area. (B) LLC and B16F10 cells grown in 10% …

https://doi.org/10.7554/eLife.37854.007
Figure 4 with 5 supplements
SCGB3A2-LPS uptake activates inflammasome signaling.

(A) Immunofluorescence analysis of LLC and ARH-77-mSDC1 cells for SCGB3A2, SDC1, and ICAM-1. Arrowheads: uropod-like structures. Cells were incubated in 0% FBS-RPMI 1640 for 40 min (0%40 m) with or …

https://doi.org/10.7554/eLife.37854.009
Figure 4—figure supplement 1
Analysis of SCGB3A2-HaloTag protein trafficking.

(A) Live cell imaging of LPSA488 binding to LLC cells. LLC cells were incubated in 1%FBS-RPMI1640 with LPSA488 (1 µg/ml) for 16 hr with HaloTag control vector-transfected supernatant (SN) or mouse …

https://doi.org/10.7554/eLife.37854.010
Figure 4—figure supplement 2
SCGB3A2 modeling.

(A) Side-by side comparison of 1UTG (uteroglobin, SCGB1A1) structure (PDBID:1UTG) and our model (SCGB3A2). S4-A-2 shows the helix number and their relative order (2-3-1-4) similar to what is …

https://doi.org/10.7554/eLife.37854.011
Figure 4—figure supplement 3
Establishment of LLC-sh-TLR4 cells and analysis of SCGB3A2/LPS binding/incorporation.

(A) Assessment of shRNA knockdown efficiency in LLC Cells. LLC cells were transfected with sh-TLR4 plasmid using retrovirus vector and stable cell clones were prepared. Each clone was grown in 2% …

https://doi.org/10.7554/eLife.37854.012
Figure 4—figure supplement 4
Effect of SCGB3A2-LPS on RAW264.7 cells.

(A) Flow cytometric analysis for SDC1 expression on RAW264.7 cells. PE conjugated mouse SDC1 ectodomain specific antibody was used for detection of cell surface SDC1 expressions on RAW264.7 cells. (B

https://doi.org/10.7554/eLife.37854.013
Figure 4—figure supplement 5
Confirmation of caspase-11 knockdown in LLC cells in the presence of Wedelolactone or LLC-sh-casp11 cells.

(A) Immunoblots of LLC cells for caspase-11 and GAPDH after treatment with or without LPS (L, 10 ng/ml), SCGB3A2 (S, 200 ng/ml), Wedelolactone (W, 50 µM) in 2%FBS-RPMI for 3 hr. C: control. (B) Immun…

https://doi.org/10.7554/eLife.37854.014
SCGB3A2-LPS promotion of pyroptotic cell death.

(A) Representative phase contrast image of LLC cells incubated with LPS (O111:B4, 1 µg/ml) and SCGB3A2 (1 µg/ml) in 1% FBS-RPMI 1640 for 72 hr. Arrows indicate cells undergoing pyroptosis (swollen …

https://doi.org/10.7554/eLife.37854.015
Evaluation for the requirement of SDC1 and other genes for SCGB3A2-LPS effect.

(A) CCK8 assay using LLC-sh-Control and LLC-sh-SDC1 for 72 hr in 1% FBS-RPMI 1640 medium. C; control, S; human SCGB3A2 (200 ng/ml), L; LPS (O111:B4, 1 pg/ml). Averages ± SD from more than three …

https://doi.org/10.7554/eLife.37854.016

Tables

Key resources table
Reagent type (species)
Or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background
(M. musculus)
Scgb3a2-/- (C57BL/6N background)In houseKido et al., 2014
Strain, strain background
(M. musculus)
Scgb3a2fl/fl (C57BL/6N background)In houseKido et al., 2014
Strain, strain background
(M. musculus)
Ccsp-Cre;LSL-K-rasG12D
(mixed background)
Francesco
DeMayo (Baylor College of Medicine, now NIEHS)
Moghaddam et al., 2009
Cell line (M. musculus)LLC-MhiGlenn Merlino (NCI)Highly metastatic, MAP test
negative
Cell line (M. musculus)B16F10ATCCCRL-6475
Cell line (M. musculus)RAW264.7
Raymond Donnelly (FDA)MAP test negative, authenticated
by STR
Cell line (human)ARH-77
ARH-77-mSDC1
Ralph Sanderson (The University of Alabama at Birmingham)MAP test
negative, authenticated by STR
Recombinant proteinmouse SCGB3A2NCI Protein Expression CoreKurotani et al., 2008
Recombinant proteinmouse SCGB3A2Hölzel DiagnosticsCSB-BP846028MO
Recombinant proteinmouse SCGB3A2CosmoBio USACSB-EP846028MO
Recombinant proteinhuman SCGB3A2In house (APCBio Innovations)Cai et al., 2014
Antibodyanti-mouse
SCGB3A2 (rabbit polyclonal)
E. Coli expressed mature SCGB3A2 in vector pET-32a(+) used as an antigen to produce a polyclonal antibody (produced by Macromolecular Resources, Fort Collins, CO)Niimi et al., 20011:5000 for IHC
Antibodyanti-human SCGB3A2 (polyclonal)In house (APCBIo Innovations)Cai et al., 20141:1000 for WB
Antibodyanti-mouse/human SDC1Pynog W Park (Harvard Med Sch)1:200 for IHC/IF
1:1000 for WB
AntibodyPE-rat anti-mouse SDC1BD Pharmingnenclone 281.21:200 for FACS
Antibodyanti-clathrin heavy chainCell Signaling TechnologyP16631:200 for IF
Antibodyanti-EEA1 (monoclonal)Cell Signaling
Technology
C45B101:200 for IF
Antibodyanti-IL-1β (monoclonal)Cell Signaling TechnologyClone 3A61:1000 for WB
Antibodyanti-caspace-11 (monoclonal)Thermo Fisher ScientificClone 17D91:1000 for WB
1:100 for IHC/IF
Antibodyanti-ICAM1 (monoclonal)Thermo Fisher ScientificMA54071:200 for IF
Antibodyanti-caspace-1 (monoclonal)Thermo Fisher ScientificClone 5B101:1000 for WB
1:100 for IF
Antibodyanti-NLRP3 (monoclonal)AdipogenAG-20B-00141:1000 for WB
1:200 for IHC/IF
Antibodyanti-LAMP1 (monoclonal)Santa Cruzsc-177681:200 for IF
Antibodyanti-HaloTag (polyclonal)PromegaG92811:100 for IF
1:1000 for WB
Antibodyanti-GAPDH (monoclonal)Proteintech60004–1-Ig1:5000 for WB
Antibodyanti-LPS (monoclonal)Abcamab356541:1000 for WB
Chemical compound, drugLPS from E. coli O111:B4Sigma-AldrichL4391
Chemical compound, drugRa mutant LPS from E.coli EH-100Sigma-AldrichL9641
Chemical compound, drugLPS from E.coli K-235Sigma-AldrichL2018
Chemical compound, drugLPS from Salmonella typhimuriumSigma-AldrichL2262
Chemical compound, drugheparin sodium salt from porcine intestinal mucosaSigma-AldrichH3393
Chemical compound, drugimidazoleSigma-AldrichI5513
Chemical compound, drugzinc sulfate solutionSigma-AldrichZ2876
Chemical compound, drugDynasoreAdooQ BioscienA12726
Chemical compound, drugWedelolactoneAdooQ BioscienA14804
Chemical compound, drugLPS-EB BiotinInvivoGentlrl-3blps

Additional files

Supplementary file 1

LPS contents in various preparations/batches of recombinant SCGB3A2

https://doi.org/10.7554/eLife.37854.017
Supplementary file 2

116 proteins identified as those significantly bound to human SCGB3A2 in protein array.

Proteins with Z-Factor >0.9, Z-Score >4.0 are listed. High score indicates a strong protein-protein interaction.

https://doi.org/10.7554/eLife.37854.018
Supplementary file 3

SCGB3A2 candidate receptors identified by human protein interaction array

https://doi.org/10.7554/eLife.37854.019
Transparent reporting form
https://doi.org/10.7554/eLife.37854.020

Download links