Impact of Hedgehog modulators on signaling pathways in primary murine and human hepatocytes in vitro: insights into liver metabolism

Abstract

The Hedgehog (Hh) signaling pathway is essential for maintaining homeostasis during embryogenesis and in adult tissues. In the liver, dysregulation of this pathway often leads to liver cancer development. Recent studies also suggest that disturbances in the Hh pathway can affect liver metabolism in healthy livers through interactions with other signaling pathways, such as the Wnt/β-catenin pathway. As a result, the Hh pathway has emerged as a promising target for therapeutic intervention. However, little is known about the effects of Hh modulators on healthy hepatocytes. In our study, we investigated the effects of the Hh agonists SAG (300 nM) and triamcinolone acetonide (40 µM), as well as the antagonists RU-SKI 43 (100 nM), cyclopamine (5 µM), budesonide (25 µM), GANT61 (0.5 µM), and vismodegib (1 µM) on healthy mouse and human primary hepatocytes in vitro. We employed toxicological, transcriptomic, proteomic, and functional assays, including proliferation and Seahorse assays. Our results show that these compounds significantly impact metabolic pathways such as lipid and glucose metabolism at both transcriptional and protein levels. Mechanistically, our data suggest the involvement of both canonical and non-canonical Hedgehog pathways, a phenomenon not previously described in hepatocytes. These findings highlight the diverse effects of these compounds on signaling and key metabolic functions in the liver, which emphasizes the need to investigate the hepatic Hh cascade and its metabolic control in depth. As the compounds regulate different aspects of metabolism, they need to be carefully studied in appropriate model systems for specific therapeutic use.

Keywords: Activator; Hedgehog signaling; Hepatocytes; Human; Inhibitor; Modulator; Murine.

Fig. 1

Principal component analysis (PCA) of a RNA and b Protein data. Hh activators (pink) and Hh inhibitors (blue) are plotted on dimensions 1 and 2. c Correlation analysis of RNA vs. protein data. p-values * ≤ 0.05; ** ≤ 0.01; *** ≤ 0.001

Fig. 2

Effect of modulators on Hh and Wnt signaling pathway-related gene and protein expression in primary hepatocytes isolated from C57BL/6N mice. qPCR analysis of a Hh and b Wnt pathway-associated gene expression in primary hepatocytes incubated with Hh modulators for 48 h (left). mRNA expression is normalized to reference gene expression and shown as log(2) fold change. N = 3–4, n = 2. Paired t-test; p-values * ≤ 0.05, ** ≤ 0.01 (p-values are available in Sup. Table S3). IPA comparison analysis with integrated fold changes from RNA-seq and proteomic analysis for (c) ‘Hh on state’ and d ‘Wnt/β-catenin signaling’ pathways. Blocks indicate measured gene expression (pink/grey) or predicted regulation of gene expression and pathway outcome (orange/blue) for genes and proteins. A full list of gene and protein expression fold changes is available in Sup. Tables S4, S5. N = 3 (pooled), n = 1

Fig. 3

Canonical pathway regulation of primary murine hepatocytes. Regulation of canonical pathways of hepatocyte-relevant functions (Lipid, amino acid, xenobiotic, and carbohydrate metabolism, autophagy, cell cycle) is depicted as treemaps with positive (orange) or negative (blue) Z-score predicted by IPA based on transcriptomic and proteomic data. Canonical pathway annotation (pathway 1–64) is available in Sup. Fig. S7, log(2) Z-scores are available in Sup. Table S6

Fig. 4

Summary of Hh modulator impact on hepatocytes. Hh modulator action on healthy hepatocytes was analyzed by Z-score (see Supplementary Tab. S6), with the most affected pathways shown here. Modulators were grouped based on similar effects on metabolism and examples of metabolic dysfunction in disease are depicted below