Krutsenko, Yekaterina
(2025)
Hepatobiliary Implications of Acute Loss of Adherens Junctions from Cholangiocytes.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Cholangiocytes, a heterogeneous population of polarized epithelial cells lining the bile ducts, play critical roles in liver function. Many hepatic pathologies stem from or are associated with disruptions of epithelial polarity and barrier integrity. Despite their importance, cholangiocyte barriers remain poorly understood. While tight junctions, the primary occluding structures, are increasingly acknowledged in liver pathobiology, adherens junctions (AJs) remain largely unexplored, especially in cholangiocytes, despite their crucial roles in adhesion, polarity, and partial contribution to barrier function. β-catenin, a well-characterized transcriptional co-activator in the liver, also functions within AJs, where its loss can be compensated by γ-catenin, a homologous desmosomal protein.
To investigate the consequences of AJ loss, we generated Opn-iCreERT2; Ctnnb1-fl/fl; Jup-fl/fl mice (DKO) to inducibly and specifically delete β- and γ-catenins in cholangiocytes. Following recombination, DKO mice exhibited 100% mortality within 5 weeks, along with severe morbidity characterized by jaundice, weight loss, lethargy, and severe liver injury. Histological analysis revealed biliary infarcts, inflammation, portal fibrosis, and stellate cell activation. CK19-positive bile ducts in DKO livers were irregular, with collapsed or absent lumens, as observed via immunohistochemistry and TEM. Severe intrahepatic cholestasis in DKO mice was evidenced by a tenfold reduction in bile flow rate and elevated bile acid levels in serum and liver. Ink injection studies demonstrated blebbing and bile leakages along the biliary tree. RT-qPCR revealed adaptive changes in bile synthesis, transport, and composition, but these were insufficient to counteract the injury. Single-nucleus RNA sequencing (snRNA-seq) identified altered hepatocyte clustering and a distinct cholangiocyte population, with gene signatures indicating junctional remodeling and upregulated necroptosis. To explore potential repair mechanisms, we induced partial AJ loss with only limited tamoxifen dosing. These mice showed improved survival (55% probability at 6 weeks), with severe hepatobiliary injury at two weeks, but significant recovery by four, and near-complete injury resolution by six weeks post recombination. The presence of A6-positive hepatocytes at a 2-week timepoint suggested hepatobiliary transdifferentiation, potentially contributing to repair.
This work highlights the essential role of AJs in cholangiocyte function and bile duct integrity. Furthermore, our model also provides a unique tool to study various aspects of cholestatic disease pathogenesis.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
13 February 2025 |
| Date Type: |
Publication |
| Defense Date: |
6 December 2024 |
| Approval Date: |
13 February 2025 |
| Submission Date: |
16 December 2024 |
| Access Restriction: |
2 year -- Restrict access to University of Pittsburgh for a period of 2 years. |
| Number of Pages: |
123 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Cellular and Molecular Pathology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
adherens junctions, beta-catenin, gamma-catenin, liver, cholangiocytes, cholestasis, cell adhesion, cell polarity, bile ducts |
| Date Deposited: |
13 Feb 2025 21:16 |
| Last Modified: |
13 Feb 2025 21:16 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/47292 |
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