Original Research
Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier Integrity in Patients With Crohn’s Disease

https://doi.org/10.1016/j.jcmgh.2020.06.005Get rights and content
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Background & Aims

Intestinal epithelial cell (IEC) barrier dysfunction is critical to the development of Crohn’s disease (CD). However, the mechanism is understudied. We recently reported increased microRNA-31-5p (miR-31-5p) expression in colonic IECs of CD patients, but downstream targets and functional consequences are unknown.

Methods

microRNA-31-5p target genes were identified by integrative analysis of RNA- and small RNA-sequencing data from colonic mucosa and confirmed by quantitative polymerase chain reaction in colonic IECs. Functional characterization of activin receptor-like kinase 1 (ACVRL1 or ALK1) in IECs was performed ex vivo using 2-dimensional cultured human primary colonic IECs. The impact of altered colonic ALK1 signaling in CD for the risk of surgery and endoscopic relapse was evaluated by a multivariate regression analysis and a Kaplan–Meier estimator.

Results

ALK1 was identified as a target of miR-31-5p in colonic IECs of CD patients and confirmed using a 3’-untranslated region reporter assay. Activation of ALK1 restricted the proliferation of colonic IECs in a 5-ethynyl-2-deoxyuridine proliferation assay and down-regulated the expression of stemness-related genes. Activated ALK1 signaling increased colonic IEC differentiation toward colonocytes. Down-regulated ALK1 signaling was associated with increased stemness and decreased colonocyte-specific marker expression in colonic IECs of CD patients compared with healthy controls. Activation of ALK1 enhanced epithelial barrier integrity in a transepithelial electrical resistance permeability assay. Lower colonic ALK1 expression was identified as an independent risk factor for surgery and was associated with a higher risk of endoscopic relapse in CD patients.

Conclusions

Decreased colonic ALK1 disrupted colonic IEC barrier integrity and was associated with poor clinical outcomes in CD patients.

Keywords

Inflammatory Bowel Disease
miR-31
ALK1
Intestinal Epithelial Barrier

Abbreviations used in this paper

BMP
bone morphogenetic protein
CD
Crohn’s disease
DM
differentiation medium
EdU
5-ethynyl-2-deoxyuridine
EM
expansion media
E2F2
E2F Transcription Factor 2
IEC
intestinal epithelial cell
HBSS
Hank’s balanced salt solution
miRNA
microRNA
mRNA
messenger RNA
NIBD
noninflammatory bowel disease
qPCR
quantitative polymerase chain reaction
SES-CD
Simple Endoscopic Score for Crohn's Disease
TEER
transepithelial electrical resistance
TGF-β
transforming growth factor β
2D
2-dimensional
UTR
untranslated region

Cited by (0)

Conflicts of interest These authors disclose the following: Nancy L. Allbritton and Yuli Wang have a financial interest in Altis Biosystems. The remaining authors disclose no conflicts.

CRediT Authorship Contributions Takahiko Toyonaga, MD, PhD (Conceptualization: Lead; Formal analysis: Lead; Investigation: Lead; Validation: Lead; Visualization: Lead; Writing – original draft: Lead; Writing – review & editing: Lead); Erin C. Steinbach (Investigation: Supporting); Benjamin P Keith (Data curation: Lead; Formal analysis: Lead; Investigation: Supporting); Jasmine B Barrow (Conceptualization: Supporting; Investigation: Supporting; Resources: Supporting); Matthew R Schaner (Resources: Lead); Elisabeth A Wolber (Resources: Lead); Caroline Beasley (Resources: Lead); Jennifer Huling (Formal analysis: Supporting; Investigation: Supporting; Methodology: Lead; Visualization: Lead); Yuli Wang (Methodology: Lead); Nancy L Allbritton (Methodology: Lead; Supervision: Lead); Nicole Chaumont (Resources: Lead); Timothy S Sadiq (Resources: Lead); Mark J Koruda (Resources: Lead); Animesh Jain (Resources: Supporting); Millie D. Long (Resources: Supporting); Edward L. Barnes (Resources: Supporting); Hans H. Herfarth (Resources: Supporting); Kim L. Isaacs (Resources: Supporting); Jonathan J. Hansen (Resources: Supporting); Michael T. Shanahan (Writing – review & editing: Supporting); Reza Rahbar (Resources: Lead); Terrence S Furey (Data curation: Lead; Formal analysis: Supporting; Funding acquisition: Supporting; Project administration: Supporting; Supervision: Lead; Writing – review & editing: Lead); Praveen Sethupathy (Formal analysis: Supporting; Funding acquisition: Supporting; Project administration: Supporting; Supervision: Lead; Writing – review & editing: Lead); Shehzad Z Sheikh, MD, PhD (Conceptualization: Lead; Formal analysis: Supporting; Funding acquisition: Lead; Methodology: Supporting; Project administration: Lead; Resources: Lead; Supervision: Lead; Validation: Supporting; Writing – review & editing: Lead).

Preprint server: This manuscript was posted to bioRxiv (doi: https://doi.org/10.1101/2020.02.21.960070). Transcript profiling was as follows: nucleotide sequence data sets discussed here are available through Gene Expression Omnibus (GEO) under accession number GSE85499 (RNA-seq; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE85499) and GSE101819 (smRNA-seq; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE101819).

Funding This work was funded in part through Helmsley Charitable Trust (SHARE Project 2); National Institute of Diabetes and Digestive and Kidney Diseases grants P01 DK094779, 1R01DK104828-01A1, R01DK109559, and P30-DK034987; T32 DK007737 (E.C.S); the Thurston Arthritis Research Center and the National Institute of Allergy and Infectious Diseases Loan Repayment Program (E.C.S.); and Career Development Award (567497) and Research Fellow Award (634239) from the Crohn’s and Colitis Foundation. The University of North Carolina Translational Pathology Laboratory is supported in part by grants from the National Cancer Institute (3P30CA016086).