Unraveling the HGF/MET axis in Mallory-Denk body pathogenesis associated with liver fibrosis through single-cell transcriptomics.

Mallory-Denk bodies (MDBs) are protein aggregates commonly observed in chronic liver diseases, including liver fibrosis. However, the intrahepatic crosstalk driving MDB pathogenesis and fibrosis progression remains poorly understood. Using single-nucleus RNA sequencing (snRNA-seq), we identified significant cellular heterogeneity and a distinct hepatocyte subpopulation, termed MDB-associated hepatocytes (MAHs). MAHs were strongly correlated with hepatocellular carcinoma progression. Four hepatic stellate cell (HSC) subpopulations were defined, among which activated HSCs (aHSCs) represent a unique MDB-associated subtype. Moreover, we revealed a tightly connected axis involving MAHs, aHSCs, and Kupffer cells (KCs), which demonstrated that aberrant hepatocyte growth factor (HGF)/mesenchymal‒epithelial transition factor (MET) signaling contributes to MDB pathogenesis. Mechanistically, HGF secreted by aHSCs or KCs interacts with MET on ballooned MAHs and stimulates the HGF/MET downstream PI3K/AKT/NF-κB and STAT3 pathways via protein phosphorylation. The activated HGF/MET pathway promotes ubiquitin D (UbD) upregulation and the release of the proinflammatory cytokine TNFα which further promotes HGF transcription, establishing a positive feedback loop and contributing to MDB formation. Furthermore, aHSCs promote MDB pathogenesis by regulating STAT3 via the HGF/MET axis and increase HSC activation by stimulating TGFβ1 secretion, thereby accelerating fibrosis in 3D MDB organoid cultures. Notably, UbD deficiency (in UbD⁻/⁻ mice) suppressed HGF/MET signaling and MDB formation, leading to reduced liver fibrosis. Consistently, HGF/MET signaling was markedly elevated in human liver biopsies containing MDBs. Together, these findings provide unprecedented single-cell insights into liver cell reprogramming and intrahepatic crosstalk during MDB pathogenesis, and highlight the HGF/MET/UbD axis as a potential therapeutic target for chronic liver disease.