Liver fibrosis: 2D expression analyses and 3D spheroid modeling suggest a potential involvement of the gene TM4SF4 in fibrotic signaling and collagen deposition

Abstract

Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is a growing global health concern that can progress to advanced liver fibrosis and cirrhosis, and it is currently lacking effective pharmacological treatment. The progression of fibrosis is characterized by the sustained activation of hepatic stellate cells (HSCs) and the excessive deposition of collagen, primarily driven by the TGFβ-1 signaling pathway. Identifying novel molecular targets for fibrosis is therefore crucial for developing therapeutic strategies to stop or reverse it. This study investigated the pro fibrotic function of TM4SF4 and its relation to the TGFβ-1 pathway. Different hepatocyte cell lines (Hep3B2, HuH7 and HepG2) were used for initial analysis in a 2D culture. This was followed by investigation into a 3D di-lineage spheroid model, consisting of hepatocytes and hepatic stellate cells. We employed small interfering RNA (siRNA) to achieve specific knockdown of TM4SF4 in 3D and TGFBR2 in 2D. Our findings first demonstrated that TM4SF4 is differentially expressed across different hepatocyte cell lines and HSCs. Moreover, TM4SF4 is significantly downregulated upon TGFβ-1 incubation in the 2D model. Mechanistically, TM4SF4 expression was found to be inversely correlated with the downregulation of TGFBR2 (TGF-β1 receptor Type 2), suggesting an interaction in the signaling cascade. Most importantly, siRNA-mediated TM4SF4 knockdown in the 3D di-lineage spheroid model resulted in a marked and consistent decrease in the levels of major fibrosis markers (COL1A1). In conclusion, this study suggests that TM4SF4 may play a key role as a regulator of liver fibrosis, given its correlation with the TGF-β1 pathway and inverse relationship with TGFBR2. These findings offer novel insights into the biology of a poorly characterized gene and propose TM4SF4 as a potential modulator in liver fibrosis.