Clec3b+ portal fibroblasts as key contributors of periportal fibrosis via the KLF4/periostin axis during cholestatic liver injury [ChIP-Seq]

ABSTRACT: Periportal fibrosis is a hallmark of cholestatic liver injury, yet the cellular origins and regulatory mechanisms underlying periportal fibrogenesis remain unclear. This study aims to identify the cellular origins and master regulators of cholestatic periportal fibrosis. Single-cell RNA sequencing was performed on bile duct-ligated (BDL) mouse model. A novel Clec3b-tdTomato lineage-tracing mouse model (Clec3bCreERT2) was generated to track portal fibroblast myofibroblastic activation in both BDL and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) mouse models. Conditional deletion of Klf4 with Clec3bCreERT2 and adeno-associated virus (AAV6)-mediated KLF4 overexpression were employed to investigate the function of KLF4 in cholestatic liver diseases models. RNA sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) were used to identify KLF4 binding targets. AAV6 carrying short hairpin RNA targeting periostin (POSTN) and neutralizing antibodies were used to gain insight into the underlying mechanisms of POSTN. Single-cell analysis identified MFAP4 as a specific marker of portal fibroblasts and their myofibroblast derivatives during cholestatic liver fibrosis. Cell-fate mapping demonstrated that Clec3b+ portal fibroblasts serve as progenitors of myofibroblasts, and contribute to periportal fibrosis. KLF4 was identified as a pivotal regulator of myofibroblastic activation in portal fibroblasts, with expression inversely correlated with fibrosis severity. Mechanistically, KLF4 represses POSTN expression by binding to its promoter, while POSTN, through interaction with integrin αvβ5, activates fibrogenic signaling in Clec3b+ portal fibroblasts. This study provides new insights into the cellular and molecular mechanisms governing periportal fibrosis in cholestatic liver disease. We identify the Clec3b+ portal fibroblasts as critical contributors to periportal fibrosis and highlight the KLF4/POSTN signaling axis as a key regulatory pathway, offering potential therapeutic approaches for cholestatic liver fibrosis.

ORGANISM(S): Mus musculus

PROVIDER: GSE298651 | GEO | 2026/03/16

REPOSITORIES: GEO