Tissue substructure-specific deposition of the β3-containing laminin-332 in the biliary epithelium of human and mouse livers

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Highlights

  • Laminin subunits expressed in bile ducts are generally conserved in humans and mice.

  • The subunits of laminin-332 exhibit heterogenous expression among bile duct cells.

  • Heterogeneous laminin-332 expression is firmly related to bile duct substructures.

  • Lamb3 is dispensable for bile duct formation and function under normal conditions.

Abstract

Laminin is a family of basement membrane proteins, whose selective and spatiotemporal expression profiles are linked to their various functions in development, maintenance, and functional regulation of different tissues. In the liver, α1-and α5-containing laminin isoforms have been documented to be critically involved in the developmental process of the epithelial tissue of the bile duct. However, possible roles of other laminin isoforms in bile duct formation and function remain elusive. Here, we evaluated public single-cell RNA sequencing databases on human liver cells to reveal expression landscape of laminin genes, and found that genes for laminin-332 subunits were conjointly expressed in the EPCAM+ biliary epithelial cell population. Expression of the β3 and γ2 subunit genes was restricted to biliary epithelial cells in the liver and, remarkably, showed apparent heterogeneity among them. We confirmed the heterogeneous nature of the laminin-β3 expression in murine livers, which was firmly related to morphological substructures in the biliary epithelium. Finally, we generated the liver epithelial tissue-specific laminin- β3 knockout mice and found that this laminin subunit was dispensable under physiological conditions. Together, our present findings have identified the β3 subunit and the related laminin-332 isoform as useful markers and potentially important regulatory molecules for future understanding of pathophysiology in the hepatobiliary system.

Introduction

The liver is a central organ for metabolism and detoxification and executes a multitude of physiological functions to maintain homeostasis in the body. One of the major functions of the liver is to produce bile, an emulsifier necessary for digestion and absorption of dietary lipids in the intestine and for discharging fat-soluble chemical wastes and toxins out of the body through the gut tube. Bile is secreted from hepatocytes and transported to the duodenum through a specialized tubular tissue structure called the bile duct, which is formed by biliary epithelial cells (BECs). In addition to its primary role as the conduit system for bile excretion, the biliary epithelial tissue is also implicated in liver regeneration, in part through showing a facultative stem/progenitor cell activity to differentiate to hepatocytes under certain types of liver injury conditions [1,2]. It is thus of fundamental importance to elucidate the mechanisms for biliary epithelial tissue development, maintenance, and function for a full understanding of physiology and pathology of the liver.

In order for a tissue to function properly, it needs to acquire and maintain a specific and well-organized structure, which can be achieved through interactions with surrounding microenvironment including extracellular matrices (ECMs) [3]. One of the major components of ECMs is laminin proteins, which comprise a family of nearly 20 different isoforms and are widely distributed throughout the whole body in multicellular organisms [4]. Importantly, laminin subunits exhibit characteristic expression profiles that are regulated in cell type- and tissue-specific manners. In vitro and in vivo studies have elucidated that cellular functions among the laminin family members are quite diverged and context-dependent, ranging from cell proliferation to cell differentiation, movement and adherence [5], which together supports the idea that selective and spatiotemporally-controlled expression profiles of laminin subtypes make significant contributions to the structural and functional specification in different tissues.

Accordingly, laminins are known to play various roles in liver development and function. With regard to the bile duct, critical roles of α1-and α5-containing laminins have been well documented. Thus, in the course of biliary development, α1-containing laminin first establishes the cell type commitment and the apical-basal polarity of the epithelium, while α5-containing laminin subsequently replaces it to induce formation of mature ducts with luminal structures [6]. However, possible involvement of other laminin isoforms in the development and functions of the bile duct remains unclear.

In this study, we performed a comprehensive analysis on the expression profile of laminin genes in the liver, taking advantage of publicized single-cell RNA sequencing (scRNA-seq) databases in humans. This enabled us to identify specific and highly characteristic expression of some laminin subunits including the β3 chain, which we subsequently confirmed to be conserved also in the mouse liver by gene expression and immunohistological analyses. Finally, to address the functional significance of the β3-containing laminin expression in the hepatobiliary system in vivo, we generated the liver epithelial-specific knockout (KO) mice for the β3 gene. The results obtained through these analyses together revealed a hitherto unrecognized unique feature of a β3-containing laminin complex in the bile duct system in human and mouse livers.

Section snippets

Animal experiments and ethics statement

All animal experiments were conducted in accordance with the Guideline for the Care and Use of Laboratory Animals of the University of Tokyo, under the approval of the Institutional Animals Experiment Ethics Committee of the Institute for Quantitative Biosciences, The University of Tokyo (approval numbers 2804, 2904, 3004, 3004–1 and 3105).

Wild-type C57BL/6J mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). C57BL/6N-Lamb3tm1c(KOMP)Wtsi/H mice carrying a floxed Lamb3 allele (hereafter

Conserved and characteristic expression of laminin subunits in bile ducts in human and mouse livers

To investigate the expression profile of laminins in human livers, particularly in relation to intrahepatic bile ducts, we utilized Human Liver Cell Atlas (http://human-liver-cell-atlas.ie-freiburg.mpg.de), an online resource for scRNA-seq data on human liver cells [12] (Fig. 1A and S1A). Laminins are heterotrimeric complexes consisting of one α chain, one β chain, and one γ chain, and there are five α, four β, and three γ chain genes in humans [4]. Each and every genes encoding these 12

Discussion

In this study, we have characterized a landscape of laminin gene expression in the liver, and have demonstrated that expression of the laminin β3 subunit is highly restricted in and also heterogenous among BECs. More specifically, the β3 subunit was found to be expressed in interlobular bile ducts, but not in bile ductules, suggesting that this molecule can serve as a potentially useful marker to discriminate these two distinct substructures in the intrahepatic biliary epithelial tissue. It has

Funding

This work was supported by AMED under Grant Numbers JP19gm6210001 (TI) and JP19fk0310111 (AM); the Japan Society for the Promotion of Science KAKENHI Grants 17H05497 (TI), 17J06605 (MY), and 17K07180 (YK); a research grant from the Takeda Science Foundation (TI); and a research grant from the Daiichi Sankyo Foundation of Life Science (TI).

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank Dr. K. Kaestner for Alfp-Cre transgenic mice, Drs. Y. Okada and E. Inoue (Institute for Quantitative Biosciences, The University of Tokyo) for in vitro fertilization of cryopreserved mouse sperm, C. Koga for cell sorting. and the University of Tokyo IQB Olympus Bio-imaging Center (TOBIC) for helping with microscopy and image acquisitions. The TROMA-III developed by Dr. Rolf Kemler was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and

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