From outside to inside: How changes in ECM composition influence cholangiocytes’ behavior

Details

Supervisors

Leclercq, Isabelle ; Manco, Rita

Faculty

Faculté de pharmacie et des sciences biomédicales

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

enIntroduction & aims: The liver is a vital organ with a key role in body homeostasis and has a remarkable regenerative capacity. However, in case of chronic injuries, native hepatocytes enter replicative senescence, and cholangiocytes begin to proliferate as Ductular Reaction (DR) cells, which can eventually differentiate into hepatocytes. Increasing evidence suggests that the extracellular matrix (ECM), especially laminin, plays a pivotal role in guiding DR cell fate. However, the mechanism by which ECM proteins such as laminin influence cholangiocytes’ behavior and identity remain to be elucidated. A known process by which mechanical cues from the microenvironment are transmitted to the nucleus, altering gene expression is called Nuclear Mechanotransduction. Thus, in this study, we explored how ECM composition regulates cholangiocyte’s identity through this process to better understand the gatekeeping role of laminin on cholangiocytes phenotype. Therefore, ECM-dependent nuclear mechanotransduction emerges as a potential lever to modulate cholangiocyte plasticity and enhance hepatocyte regeneration in chronic liver disease. Methods: To be able to study the influence of ECM on cholangiocyte’s identity, we used normal mouse cholangiocytes (NMC) cultures in a double-spheroid model. This model allows to control the ECM composition, and, in this case, we compared cell behavior in laminin-rich versus collagen I-rich matrices. ECM influences on cholangiocytes’ behavior were analyzed through transcriptomic analysis, immunofluorescence, RT-qPCR techniques, imaging, flow cytometry and functional analysis. Results: Imaging and transcriptomics analysis confirmed that our double-spheroid model is robust and that laminin is the gatekeeper of cholangiocyte’s phenotype. Indeed, NMC embedded in laminin formed branch-like structure and maintained the biliary phenotype, while those embedded in collagen I, display dispersed, unstructured spreading behavior and stress-responsive features. Moreover, transcriptomics analysis showed ECM-specific signatures: laminin enriched bile secretion pathways, while collagen I triggered stress and inflammatory responses. ECM composition also affected nuclear morphology and organization. Laminin promoted smaller, rounder, stiffer nuclei and increased lamina associated domains (LADs), consistent with transcriptional stability. However, when cultured in collagen I, cells had an irregular nuclear shapes and fewer LADs, indicating higher transcriptional plasticity. Noticeably, when disrupting the Nuclear Mechanotransduction signal using Y-27632, a Rho associated coiled-coil containing protein kinase (ROCK) inhibitor, the difference observed before are lost, showing that ECM influences cell’s behavior and identity. Conclusion: Nuclear Mechanotransduction is the central mechanism by which ECM composition influence cell fate, and in this case cholangiocyte’s identity. These studies offer insight into potential targets such as ECM or nuclear mechanotransduction, to modulate change in cholangiocytes phenotypes and their differentiation in hepatocytes.