For experiments of antibody administration, Hamster anti-rat CD29 (555002, BD Pharmingen) or Hamster IgM (553957, BD Pharmingen) was added in the culture at a final concentration of 1 1 g/mL. Immunohistochemistry The resected left lobe of liver was embedded into OCT compound (Sakura Finetek Japan), and frozen by liquid nitrogen. the details of formed cyst in the 3D culture using Lu- BC in the presence or absence of neutralizing anti-Itgb1 antibody. elife-36572-fig5-data1.xlsx (40K) DOI:?10.7554/eLife.36572.023 Figure 5figure supplement 2source data 1: Numerical data for expression analysis of mRNA in Lu+ BC and Lu- BC by quantitative RT-PCR. elife-36572-fig5-figsupp2-data1.xlsx (17K) DOI:?10.7554/eLife.36572.018 Figure 5figure supplement 3source data 1: Numerical data for expression analysis of mRNA in Lu- BC by quantitative RT-PCR. elife-36572-fig5-figsupp3-data1.xlsx (15K) DOI:?10.7554/eLife.36572.020 Figure 5figure supplement 4source data 1: Numerical data for the details of formed cyst in the 3D culture using Lu+ BC in the presence or absence of activating anti-Itgb1 antibody (TS2/16). elife-36572-fig5-figsupp4-data1.xlsx (34K) DOI:?10.7554/eLife.36572.022 Figure 6source data 1: Figure 6B: Numerical data for the ratio of Ki67+ cells per EpCAM+ cells. Figure 6D: Numerical data for measurements of the distance from portal vein to distal biliary cells in the CDE and DDC models. elife-36572-fig6-data1.xlsx (57K) DOI:?10.7554/eLife.36572.026 Transparent reporting form. elife-36572-transrepform.docx (245K) DOI:?10.7554/eLife.36572.029 Data Availability StatementAll data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 3,4,5, 6 and Supporting figure 5. Abstract Under chronic or severe liver injury, liver progenitor cells (LPCs) of biliary origin are known to expand and contribute to the regeneration of hepatocytes and cholangiocytes. This regeneration process is called ductular reaction (DR), which is accompanied by dynamic remodeling of biliary tissue. Although the DR shows apparently distinct mode of biliary extension depending on the type of TNFRSF4 liver injury, the key regulatory mechanism remains poorly understood. Here, we show that Lutheran (Lu)/Basal cell adhesion molecule (BCAM) regulates the morphogenesis of DR depending on liver disease models. Lu+ and Lu- biliary cells isolated from injured liver exhibit opposite phenotypes in cell motility and duct formation Fosamprenavir Calcium Salt capacities in vitro. By overexpression of Lu, Lu- biliary cells acquire Fosamprenavir Calcium Salt the phenotype of Lu+ biliary cells. Lu-deficient mice showed severe defects in DR. Our findings reveal a critical role of Lu in the control of phenotypic heterogeneity of DR in distinct liver disease models. mRNA was verified in both EpCAM+ biliary cells isolated from CDE- and DDC-injured livers (Figure 5B), implying the involvement of Laminins in Lu-driven regulation. While Lu is capable of binding to Laminin-511/521 via Lama5, these laminins are also known as a ligand for Integrin31/61 (Kikkawa Fosamprenavir Calcium Salt et al., 2007). It has been reported that Lu binds to Lama5 competitively with Integrin31/61 and promotes tumor cell migration by modulating Integrin-mediated cell attachment to Laminin-511 protein (Kikkawa et al., 2013). Taking these evidences into account, Lu may regulate the morphogenesis of DR via an Integrin-mediated manner. Given that Lu plays a role in the competitive inhibition against Laminin-511/521 and Integrin31/61 axis in biliary cell as shown in Figure 5figure supplement 1, high expression of Lu would be reproduced by inhibition of integrin1 (Itgb1) signaling. To address this possibility, we first investigated the expression of ((in Lu- Fosamprenavir Calcium Salt BC and Lu+ BC. As shown in Figure 5figure supplement 2, all integrin components were expressed in Lu- BC and Lu+ BC, indicating that Lu- BC and Lu+ BC are potentially competent to cell signaling via Integrin31/61-Laminin-511/521 axis. We next examined the effect of neutralizing antibody against Itgb1 on the motility and duct formation capacity of Lu- BC in vitro. Although the inhibition of Itgb1 signaling did not affect the expression of Lu (Figure 5figure supplement 3), it dramatically changed Lu- BC to Lu+ BC-like phenotype in both scratch assay and cyst formation assay (Figure 5C and D). Conversely, we investigated the effect of Itgb1 activation on Lu+ BC. Because TS2/16 antibody has been reported to activate Itgb1 signaling (Rozo et al., 2016), we added it to the 3D culture of Lu+ BC. As a result, Lu+ BC acquired Fosamprenavir Calcium Salt cyst formation capacity by the activation of Itgb1 (Figure 5figure supplement 4). These data strongly suggested that Lu regulates the characteristic of DR by modulating the Itgb1 signaling. Open in a separate window Figure 5. Itgb1 signaling is critical for regulating the phenotype of biliary cells.(A) Expression analysis for Lama5 in injured liver. Co-staining of EpCAM and Lama5 was performed in liver sections of CDE-fed mouse and DDC-fed mouse. (B) Evaluation of gene expression in EpCAM+ cells isolated from CDE-fed and DDC-fed mouse livers by quantitative RT-PCR. Data are plotted in a.