Neurotrophic factor GDNF regulates intestinal barrier function in inflammatory bowel disease
Michael Meir, … , Jens Waschke, Nicolas Schlegel
Michael Meir, … , Jens Waschke, Nicolas Schlegel
Published July 1, 2019; First published June 17, 2019
Citation Information: J Clin Invest. 2019;129(7):2824-2840. https://doi.org/10.1172/JCI120261.
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Categories: Research Article Gastroenterology

Neurotrophic factor GDNF regulates intestinal barrier function in inflammatory bowel disease

Abstract

Impaired intestinal epithelial barrier (IEB) function with loss of desmosomal junctional protein desmoglein 2 (DSG2) is a hallmark in the pathogenesis of inflammatory bowel disease (IBD). While previous studies have reported that glial cell line–derived neurotrophic factor (GDNF) promotes IEB function, the mechanisms are poorly understood. We hypothesized that GDNF is involved in the loss of DSG2, resulting in impaired IEB function as seen in IBD. In the inflamed intestine of patients with IBD, there was a decrease in GDNF concentrations accompanied by a loss of DSG2, changes of the intermediate filament system, and increased phosphorylation of p38 MAPK and cytokeratins. DSG2-deficient and RET-deficient Caco2 cells revealed that GDNF specifically recruits DSG2 to the cell borders, resulting in increased DSG2-mediated intercellular adhesion via the RET receptor. Challenge of Caco2 cells and enteroids with proinflammatory cytokines as well as dextran sulfate sodium–induced (DSS-induced) colitis in C57Bl/6 mice led to impaired IEB function with reduced DSG2 mediated by p38 MAPK–dependent phosphorylation of cytokeratins. GDNF blocked all inflammation-induced changes in the IEB. GDNF attenuates inflammation-induced impairment of IEB function caused by the loss of DSG2 through p38 MAPK–dependent phosphorylation of cytokeratin. The reduced GDNF in patients with IBD indicates a disease-relevant contribution to the development of IEB dysfunction.

Authors

Michael Meir, Natalie Burkard, Hanna Ungewiß, Markus Diefenbacher, Sven Flemming, Felix Kannapin, Christoph-Thomas Germer, Matthias Schweinlin, Marco Metzger, Jens Waschke, Nicolas Schlegel

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Figure 2

GDNF stabilizes the intestinal barrier via DSG2.

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GDNF stabilizes the intestinal barrier via DSG2. (A) Immunostaining of C...
(A) Immunostaining of Caco2 monolayers at confluency for DSG2 and after application of 100 ng/ml GDNF for 24 hours; n = 10. Scale bar, 20 µm. (B) Western blot for DSG2 after application of GDNF (n = 5; unpaired t test). (C) DSG2 was augmented in the triton-insoluble fraction after GDNF application in triton extraction experiments (n = 7; Kruskal-Wallis test, ANOVA). (D) In AFM measurements, living Caco2 cell topography images were created for selection of specific areas at cell borders (left panel). These areas (white boxes) were inserted as an overlay into the image to exemplify where measurements were carried out. Force measurements with a DSG2-coated AFM cantilever revealed binding events on the surface of Caco2 cells with each white dot representing 1 binding event (right panel). (E) Analysis of measured DSG2-specific unbinding forces resulted in a distribution peak of 29.3 pN. (F) Cell topography of Caco2 cells was imaged after incubation with GDNF (left panel). Areas surrounded by white boxes were inserted as an overlay into the image to exemplify where measurements were carried out. Force maps of DSG2 adhesion measurements display more binding events along the cell border (right panel) after application of GDNF. (G) Quantification of measured binding events showed increased binding frequency after application of GDNF and decreased binding frequency after EGTA-mediated Ca2+ depletion (n = 6 for GDNF, n = 3 for EGTA; Kruskal-Wallis test, ANOVA). (H) Distribution ratio of DSG2-specific binding events was calculated as the quotient of measured binding events along the cell border (cb) and measured binding events on the surrounding cell surface (cs) (n = 6, paired t test). (I) Western blot for DSG2 to confirm the effective knock out of DSG2 in the Caco2 DSG2–/– cell line (n = 9; Kruskal-Wallis test, ANOVA). (J and K) TER measurements of Caco2 DSG2+/+ cells transfected with nonsense plasmids (J) and Caco2 DSG2–/– following application of 100 ng/ml GDNF are shown (n = 6 experiments for each condition). *P < 0.05; unpaired t tests for each time point; OD values normalized to β-actin.