Abstract

Background: Primary sclerosing cholangitis (PSC) is marked by inflammation and progressive biliary fibrosis, which can lead to cirrhosis and its complications. Cholangiocytes activated by transforming growth factor-𝛽 (TGF𝛽) signal to immune cells and activate hepatic myofibroblasts to deposit the extracellular matrix. Our previous data suggest that TGF𝛽-mediated transcriptomic changes in cholangiocytes may occur through runt-related transcription factors (RUNX). However, studies of RUNX1 in hepatobiliary fibrosis have revealed conflicting findings because of unexplored mechanistic understanding in cholangiocytes, which is the focus of this study.

Methods: Mouse large biliary epithelial cells (MLE) and PSC-derived cholangiocytes (PSC-C) were used to test the effects of RUNX inhibitors (Ro5-3335 and AI-10-104) and siRNA knockdown on TGF𝛽-mediated signaling. Multidrug resistance 2 deleted (Mdr2^-/-) mice (12 weeks, male and female) were treated with the RUNX inhibitor Ro5-3335 intraperitoneally at 50 mg/kg every other day for 3 weeks.

Results: RUNX1 mRNA is significantly increased in TGF𝛽-treated cholangiocytes, Mdr2^-/- mouse cholangiocytes and RNA-seq of PSC tissue (Log Fc 1.63) (GEO data set: GSE159676). RUNX inhibitors significantly reduced the expression of fibroinflammatory markers such as platelet-derived growth factor B (PDGFB) and interleukin 6 (IL-6) in TGF𝛽 treated MLE. Ro5-3335, also reduced the basal expression of PDGFB and IL-6 in PSC-C. RUNX1 specific siRNA knockdown in PSC-C reduced the basal expression of IL-6. Conversely, the expression of anti-inflammatory and anti-fibrotic, peroxisome proliferator-activated receptor 𝛾 (PPAR𝛾) was increased. Mdr2^-/- mice treated with Ro5-3335 showed significant reductions in serum alanine transaminase and hepatic expression of inflammatory markers (Il-6, Tnfa, Il-1b, Nfkb) by 40-75% but not the anti-inflammatory cytokine, Il-10. In contrast, mRNA markers (Collagen, ⍺-Smooth muscle actin) and picrosirius red histological staining of fibrosis did not show a significant reduction.

Conclusion: RUNX1 has an essential role in TGF𝛽-mediated activation of the inflammatory response in cholangiocytes and Mdr2^-/- mice. We are conducting longer in vivo experiments of RUNX inhibition to determine the effects on biliary fibrosis. Cholangiocyte-selective RUNX1 knockout mice will also be used for further investigation. Targeting RUNX1 may represent a novel therapeutic strategy in cholestatic liver disease.