The ESRP1-GPR137 axis contributes to intestinal pathogenesis

  1. Lukas Franz Mager
  2. Viktor Hendrik Koelzer
  3. Regula Stuber
  4. Lester Thoo
  5. Irene Keller
  6. Ivonne Koeck
  7. Maya Langenegger
  8. Cedric Simillion
  9. Simona P Pfister
  10. Martin Faderl
  11. Vera Genitsch
  12. Irina Tcymbarevich
  13. Pascal Juillerat
  14. Xiaohong Li
  15. Yu Xia
  16. Eva Karamitopoulou
  17. Ruth Lyck
  18. Inti Zlobec
  19. Siegfried Hapfelmeier
  20. Rémy Bruggmann
  21. Kathy D McCoy
  22. Andrew J Macpherson
  23. Christoph Müller
  24. Bruce Beutler
  25. Philippe Krebs  Is a corresponding author
  1. University of Bern, Switzerland
  2. University Hospital Zurich, Switzerland
  3. Inselspital, Bern University Hospital, University of Bern, Switzerland
  4. University of Texas Southwestern Medical Center, United States
  5. The Scripps Research Institute, United States
11 figures, 3 tables and 3 additional files

Figures

Figure 1 with 1 supplement
Esrp1Triaka leads to altered mRNA splicing and reduced epithelial cell proliferation.

To quantify exon splicing, HEK-293 cells were co-transfected with a vector encoding Esrp1WT or Esrp1Triaka or an empty control (CTRL) vector and with an exon trap construct containing (A) Cd44

https://doi.org/10.7554/eLife.28366.003
Figure 1—figure supplement 1
Mapping of the Triaka mutation.

(A) The Triaka mutation was mapped based on the hyperactivity and circling phenotype of the strain to proximal Chromosome 4, with a peak LOD score of 2.71 at D4Mit235. (B) The Triaka mutation causes …

https://doi.org/10.7554/eLife.28366.004
Figure 2 with 1 supplement
Esrp1Triaka alters mRNA splicing patterns in colonic intestinal epithelial cells.

(A) RNA sequencing analysis was performed on colonic intestinal epithelial cells (cIECs) isolated from WT and Triaka mice. Dot plot indicating the relative difference in isoform usage for a given …

https://doi.org/10.7554/eLife.28366.005
Figure 2—source data 1

Altered ratios of transcript isoforms in Triaka epithelial cells.

https://doi.org/10.7554/eLife.28366.007
Figure 2—figure supplement 1
CD44 expression in Triaka and WT colons.

Immunohistochemistry was performed on colonic tissue of indicated mice to detect total CD44 (CD44t) or CD44v4. Representative pictures are shown. Scale bars: 50 µm (magnification of data shown in Fig…

https://doi.org/10.7554/eLife.28366.006
Figure 3 with 3 supplements
Esrp1Triaka decreases the barrier function of the intestine.

(A) Representative flow cytometry plot of colonic single cells stained for EpCAM and CD45 (left panel). Representative histogram indicating surface E-cadherin expression on EpCAM+ epithelial cells …

https://doi.org/10.7554/eLife.28366.008
Figure 3—source data 1

Gene expression and pathway analysis.

https://doi.org/10.7554/eLife.28366.012
Figure 3—figure supplement 1
Esrp1Triaka alters the expression of genes associated with cell proliferation.

RNA sequencing data were used to perform a gene set enrichment analysis using the SetRank method. LogFC plots show genes that are differently expressed in Triaka versus WT colonic intestinal …

https://doi.org/10.7554/eLife.28366.009
Figure 3—figure supplement 2
Normal colonic histomorphology in Triaka mice.

Intestinal sections of WT and Triaka mice. (A) Representative H&E-stained sections of the large intestine. (B) Colonic crypt depth was measured on H&E stained sections (n = 6 mice per group). (C) …

https://doi.org/10.7554/eLife.28366.010
Figure 3—figure supplement 3
Normal small intestinal barrier integrity, fecal lipocalin-2 and albumin levels in Triaka mice.

(A) Barrier resistance in the small intestine tissue of WT and Triaka was measured using an Ussing chamber (n = 6 mice per group). Fecal pellets of WT and Triaka animals were collected to measure …

https://doi.org/10.7554/eLife.28366.011
Figure 4 with 3 supplements
Esrp1Triaka modulates the course of experimental intestinal immunopathology.

(A) WT (n = 7) and Triaka (n = 4) mice were challenged with 2% DSS in the drinking water for 7 days. Weight loss was measured daily (left panel). One representative out of four different experiments …

https://doi.org/10.7554/eLife.28366.014
Figure 4—figure supplement 1
Triaka mice show increased susceptibility to experimental colitis.

(A) WT and Triaka mice were treated with 2% DSS water and disease activity score was assessed at the indicated time points (n = 4–7 mice per group). (B) Colitis score was assessed by histological …

https://doi.org/10.7554/eLife.28366.015
Figure 4—figure supplement 2
More aggressive molecular signature in Triaka intestinal tumors.

WT and Triaka mice were treated with AOM/DSS. Seventy days after the first AOM injection, tumors or adjacent tumor-free (healthy) colonic tissue were homogenized and the indicated proteins were …

https://doi.org/10.7554/eLife.28366.016
Figure 4—figure supplement 3
Partial EMT signature in Triaka cIECs.

Quantitative PCR was applied to measure transcript levels of (A) Cdh1, (B) Zeb1, (C) Zeb2, and (D) Esrp1 in WT and Triaka cIECs, after normalization to Gapdh expression. n = 9–14 mice per group. …

https://doi.org/10.7554/eLife.28366.017
Figure 5 with 3 supplements
GPR137 isoforms differentially activate Wnt/β-catenin signaling to modulate epithelial cell function.

(A) CMT-93 IECs were transduced with vectors encoding the indicated Gpr137 isoforms or a control vector (CTRL) and live cells were counted daily by microscopy. (B) Alternatively, monolayer …

https://doi.org/10.7554/eLife.28366.018
Figure 5—figure supplement 1
Gpr137 is a splicing target of ESRP1.

To quantify exon splicing, HEK-293 cells were co-transfected with a vector encoding Esrp1WT or Esrp1Triaka or an empty control (CTRL) vector and with an exon trap construct containing Gpr137 exon 2 …

https://doi.org/10.7554/eLife.28366.019
Figure 5—figure supplement 2
Gpr137 isoforms differently modulate the proliferation of epithelial cells.

CMT-93 IECs were transduced with vectors encoding the indicated Gpr137 isoforms and (A) cell proliferation relative to control vector-transduced cells was measured in a WST-1 assay. (B) Transcript …

https://doi.org/10.7554/eLife.28366.020
Figure 5—figure supplement 3
Reduced Wnt/β-catenin signaling in Esrp1Triaka- compared with Esrp1WT-transduced cells.

CMT-93 cells were transduced with inducible lentiviral vectors encoding Esrp1Triaka or Esrp1WT and active β-catenin was measured by flow cytometry, after treatment with 4-hydroxytamoxifen. …

https://doi.org/10.7554/eLife.28366.021
Figure 6 with 2 supplements
Expression of ESRP1 and ESRP1-dependent hGPR137 isoforms is down-regulated in the diseased intestine and predicts CRC patient survival.

(A) ESRP1 transcript levels were measured in inflamed versus matched, non-inflamed intestinal biopsies from Crohn’s disease (CD) patients and normalized to EPCAM expression. Normalized ESRP1

https://doi.org/10.7554/eLife.28366.022
Figure 6—source data 1

Association of (nuclear) ESRP1 expression with clinicopathological features in 185 CRC patients.

https://doi.org/10.7554/eLife.28366.025
Figure 6—source data 2

Univariate and multivariate survival analysis in 185 CRC patients.

https://doi.org/10.7554/eLife.28366.026
Figure 6—source data 3

Correlation of hGPR137_Short with Wnt target genes.

https://doi.org/10.7554/eLife.28366.027
Figure 6—figure supplement 1
Expression of GPR137 isoforms in the healthy and diseased intestine.

(A) Transcript levels of ESRP1 and hGPR137_Short were measured in normal human colon tissue (n = 10) and normalized to GAPDH expression. Expression of hGPR137_Short was assessed using primers …

https://doi.org/10.7554/eLife.28366.023
Figure 6—figure supplement 2
ESRP1-dependent alternative mRNA splicing is required for epithelial integrity and intestinal homeostasis.

(A) (1) The Esrp1Triaka allele generates altered mRNA splicing events. (2) Perturbation in the distribution of specific isoforms or generation of aberrant protein isoforms in epithelial cells reduce …

https://doi.org/10.7554/eLife.28366.024
Author response image 1
Expression analysis of selected genes with known function for intestinal barrier integrity.

(A) RNA sequencing was performed on colonic intestinal epithelial cells (cIECs) from Triaka and WT mice to assess expression of the indicated genes (n = 4 mice per group). (B) Quantitative PCR was …

https://doi.org/10.7554/eLife.28366.033
Author response image 2
Expression analysis of selected genes with known function for post-translational modification of intestinal mucus.

RNA sequencing was performed on colonic intestinal epithelial cells (cIECs) from Triaka and WT mice (n = 4 mice per group) to assess expression of the indicated genes involved in (A) glycosylation …

https://doi.org/10.7554/eLife.28366.034
Author response image 3
Analysis Mucin-2 (MUC2) layer.

(A) Immunofluorescence for MUC2 (in green) was performed on colon tissue of the indicated strains. Nuclei were visualized with DAPI. Representative pictures are shown (scale bar: 100µm). (B) …

https://doi.org/10.7554/eLife.28366.036
Author response image 4
Partial EMT signature in Triaka cIECs.

(A) RNA sequencing was performed on colonic intestinal epithelial cells (cIECs) from Triaka and WT mice to assess expression of the indicated epithelial or mesenchymal marker genes (n = 4 mice per …

https://doi.org/10.7554/eLife.28366.037
Author response image 5
Availability of target transcripts may determine the effect of the Esrp1Triaka mutation.

Fgfr2-IIIb expression levels were measured in EPCAM+-sorted colonic intestinal epithelial cells of the indicated strains and normalized to Gapdh expression.

Intestinal epithelial cells were isolated from mice at steady-state or from animals that underwent a short 3 day-treatment with dextran sodium sulfate (indicated as “Inflammatory conditions”) (n = 9 mice per group for steady-state conditions and n = 7 mice per group during inflammation conditions. Statistics: (B) Mann-Whitney test. *, P < 0.05.

https://doi.org/10.7554/eLife.28366.038

Tables

Table 1
Bacterial translocation and serum anti-commensal antibodies.
https://doi.org/10.7554/eLife.28366.013
No. of WT miceNo. of Triaka micep-value
Penetration of bacteria in mucus or mucosa
16S rRNA0/96/80.0023
Serum anti-commensal antibodies
IgG11/107/100.0198
IgG2b1/108/100.0055
  1. Statistics: Fisher's exact test was performed. This table relates to Figure 3.

Table 2
Gradual loss of nuclear ESRP1 expression is associated with CRC progression
https://doi.org/10.7554/eLife.28366.028
Tissue typeNumber of casesESRP1 expression (%)p-value
AverageMedian
Normal267575<0.0001
Adenoma4256.860
Carcinoma18526.515
Lymph node metastasis689.10
  1. Statistics: Kruskal-Wallis test was performed. This table relates to Figure 6.

Author response table 1
Unaltered barrier function or mucus production/modification pathways in Triaka cIECs.
https://doi.org/10.7554/eLife.28366.035
SetIDDatabaseDescriptionChange Triaka vs. WT
GO:0070254GOBPmucus secretionNot significant
GO:0070255GOBPregulation of mucus secretionNot significant
GO:0070256GOBPnegative regulation of mucus secretionNot significant
GO:0070257GOBPpositive regulation of mucus secretionNot significant
GO:0070701GOCCmucus layerNot significant
GO:0070702GOCCinner mucus layerNot significant
GO:0070703GOCCouter mucus layerNot significant
GO:0006486GOBPprotein glycosylationNot significant
GO:0006487GOBPprotein N-linked glycosylationNot significant
GO:0006493GOBPprotein O-linked glycosylationNot significant
GO:0006517GOBPprotein deglycosylationNot significant
GO:0018242GOBPprotein O-linked glycosylation via serineNot significant
GO:0018243GOBPprotein O-linked glycosylation via threonineNot significant
GO:0018279GOBPprotein N-linked glycosylation via asparagineNot significant
GO:0033575GOBPprotein glycosylation at cell surfaceNot significant
GO:0033577GOBPprotein glycosylation in endoplasmic reticulumNot significant
GO:0033578GOBPprotein glycosylation in GolgiNot significant
GO:0060049GOBPregulation of protein glycosylationNot significant
GO:0060050GOBPpositive regulation of protein glycosylationNot significant
GO:0060051GOBPnegative regulation of protein glycosylationNot significant
GO:0090283GOBPregulation of protein glycosylation in GolgiNot significant
GO:0090284GOBPpositive regulation of protein glycosylation in GolgiNot significant
GO:0090285GOBPnegative regulation of protein glycosylation in GolgiNot significant
5894152ReactomeO-glycosylation of TSR domain-containing proteinsNot significant
GO:0070830GOBPtight junction assemblyNot significant
GO:1902396GOBPprotein localization to tight junctionNot significant
GO:2000810GOBPregulation of tight junction assemblyNot significant
GO:0005923GOCCtight junctionNot significant
GO:0007045GOBPcell-substrate adherens junction assemblyNot significant
GO:0034332GOBPadherens junction organizationNot significant
GO:0034333GOBPadherens junction assemblyNot significant
GO:0034334GOBPadherens junction maintenanceNot significant
GO:0071896GOBPprotein localization to adherens junctionNot significant
GO:0005912GOCCadherens junctionNot significant
GO:0005913GOCCcell-cell adherens junctionNot significant
GO:0005914GOCCspot adherens junctionNot significant
GO:0005924GOCCcell-substrate adherens junctionNot significant

Additional files

Supplementary file 1

Antibodies and conjugates used for flow cytometry analysis.

https://doi.org/10.7554/eLife.28366.029
Supplementary file 2

Self-designed primers for qPCR analysis.

https://doi.org/10.7554/eLife.28366.030
Transparent reporting form
https://doi.org/10.7554/eLife.28366.031

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