Gastroenterology

Gastroenterology

Volume 161, Issue 6, December 2021, Pages 1924-1939
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
The Tight Junction Protein ZO-1 Is Dispensable for Barrier Function but Critical for Effective Mucosal Repair

https://doi.org/10.1053/j.gastro.2021.08.047Get rights and content

Backgrounds & Aims

Increased permeability is implicated in the pathogenesis of intestinal disease. In vitro and in vivo studies have linked down-regulation of the scaffolding protein ZO-1, encoded by the TJP1 gene, to increased tight junction permeability. This has not, however, been tested in vivo. Here, we assessed the contributions of ZO-1 to in vivo epithelial barrier function and mucosal homeostasis.

Methods

Public Gene Expression Omnibus data sets and biopsy specimens from patients with inflammatory bowel disease (IBD) and healthy control individuals were analyzed. Tjp1f/f;vil-CreTg mice with intestinal epithelial–specific ZO-1 knockout (ZO-1KO.IEC) mice and Tjp1f/f mice littermates without Cre expression were studied using chemical and immune-mediated models of disease as well as colonic stem cell cultures.

Results

ZO-1 transcript and protein expression were reduced in biopsy specimens from patients with IBD. Despite mildly increased intestinal permeability, ZO-1KO.IEC mice were healthy and did not develop spontaneous disease. ZO-1KO.IEC mice were, however, hypersensitive to mucosal insults and displayed defective repair. Furthermore, ZO-1–deficient colonic epithelia failed to up-regulate proliferation in response to damage in vivo or Wnt signaling in vitro. ZO-1 was associated with centrioles in interphase cells and mitotic spindle poles during division. In the absence of ZO-1, mitotic spindles failed to correctly orient, resulting in mitotic catastrophe and abortive proliferation. ZO-1 is, therefore, critical for up-regulation of epithelial proliferation and successful completion of mitosis.

Conclusions

ZO-1 makes critical, tight junction–independent contributions to Wnt signaling and mitotic spindle orientation. As a result, ZO-1 is essential for mucosal repair. We speculate that ZO-1 down-regulation may be one cause of ineffective mucosal healing in patients with IBD.

Section snippets

Materials and Methods

Mouse studies followed protocols approved by institutional animal care and use committees and used vil-mRFP1-ZO-1 (Tg(Vil1-mRFP1/TJP1)#Tjr, MGI:5584023), Tjp1f/f (Tjp1tm2c(KOMP)Wtsi, MGI:6272009), vil-Cre (B6.Cg-Tg(Vil1-cre)20Syr, MGI:3053819), vil-CreERT2 (B6.Cg-Tg(Vil1-cre/ERT2)23Syr/J, MGI:6278020), H2B-mCherry (R26-H2B-mCherry, CDB:CDB0239K), and GFP-β-actin (Pfn1tm2.1(GFP/ACTB)Wit, MGI:5568700) mice. Tjp1f/f littermates were used as wild-type (WT) controls for Tjp1f/f-vil-Cre (ZO-1KO.IEC)

ZO-1 Expression Is Down-regulated in Inflammatory Bowel Disease

To comprehensively assess tight junction protein transcription in IBD, we screened the GDS3268 RNA microarray data set, which includes biopsy specimens from 67 patients with ulcerative colitis and 31 healthy control individuals.8 This confirmed published results showing increased CLDN1 and CLDN2 and reduced OCLN, CLDN4, and CLDN8 expression in ulcerative colitis (Figure 1A).6,7 These changes were generally consistent across 5 additional ulcerative colitis and 4 Crohn’s disease data sets (

Discussion

ZO-1 was the first epithelial tight junction protein identified,24 but it is also expressed in cells that do not form tight junctions, including blastomeres at the 8-cell stage of mammalian development.25 This contrasts with some other tight junction proteins whose expression is limited to epithelial and endothelial cells and whose knockout induces only subtle abnormalities.26, 27, 28 In contrast, knockout of Tjp1, which encodes ZO-1, leads to the developmental defects as early as embryonic day

Acknowledgments

The authors thank Tiffany S. Davanzo (Slaybaugh Studios) for her beautiful illustrations and Heather Marlatt (Nationwide Histology) for her outstanding assistance with histologic staining and tissue microarray development.

Wei Ting Kuo’s current affiliation is the Graduate Institute of Oral Biology, National Taiwan University, Taipei, Taiwan. Gurminder Singh’s current affiliation is AbbVie Inc, Cambridge, Massachusetts.

CRediT Authorship Contributions

Wei-Ting Kuo, PhD (Conceptualization: Lead; Investigation: Lead; Writing –

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    Conflicts of interest Guminder Singh is an employee of AbbVie, Inc. Jerrold R. Turner is a founder and shareholder of Thelium Therapeutics and has served as a consultant for Entrinsic, Immunic, and Kallyope. The remaining authors disclose no conflicts.

    Funding This work was supported by National Institutes of Health grants R01DK61931 (to Jerrold R. Turner), R01DK68271 (to Jerrold R. Turner), R01DK099097 (to Clara Abraham), and P30DK034854 (to the Harvard Digestive Disease Center) and by the National Natural Science Foundation of China grants 81800464 (to Li Zuo) and 82070548 (to Li Zuo).

    Author names in bold designate shared co-first authorship.

    This article has an accompanying continuing medical education activity, also eligible for MOC credit, on page e18. Learning Objective: Upon completion of this CME activity, successful learners will recognize that, despite being the first tight junction protein discovered, ZO-1 is not required for tight junction barrier function. They will also be able to explain how changes in intestinal epithelial ZO-1 expression is associated with colitis, identify the two noncanonical mechanisms by which ZO-1 promotes mucosal repair, and explain the hypothesis that ZO-1 loss contributes to defective mucosal healing in inflammatory bowel disease.

    Authors share co-first authorship.

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