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Butyrylcholinesterase alleviates hepatic stellate cell activation via paracrine muscarinic signaling [2W-CCL4-DOX-HSC]

ABSTRACT: Background & Aims: Butyrylcholinesterase (BuChE) is an enzyme involved in Alzheimer’s disease (AD), detoxification and depression. While BuChE expression is reduced in liver cirrhosis, its physiological substrate, acetylcholine (ACh), promotes fibrogenic responses in hepatic stellate cells (HSCs). However, the function of BuChE in liver fibrosis and mechanistic link between these two observations is not known. Methods: BuChE expression and HSC activation were assessed in liver biopsies from patients with liver fibrosis using qPCR and immunostaining. To investigate the functional role of BuChE in fibrogenesis, we employed BucheΔHep, knock-in (BucheKI/KI-Hep) mice, as well as pharmacological inhibition. The inhibitory effect of BuChE on HSC activation was further confirmed using conditioned medium from cells expressing mutant BuChE, in hepatocyte-HSC co-culture experiments, and analyses of recombinant BuChE protein. The release of ACh was measured with patch-clamp and the expression and functionality of its receptor were detected via intracellular Ca2+ influx measurements. The role of the muscarinic acetylcholine receptor 3 (ChRM3) in HSCs was investigated through gain- and loss-of-function experiments, utilizing Chrm3ΔHSC mice and AAV-mediated Chrm3 overexpression driven by the alpha-smooth muscle actin (Acta2) promoter. Results: Buche mRNA expression in livers and enzymatic activity in serum are downregulated in patients and mouse models with liver fibrosis, and show a negative correlation with F stage and ACTA2 expression. Pharmacological inhibition or depletion of BuChE in hepatocytes results in increased HSC activation and aggravates liver fibrosis in various mouse models, whereas its overexpression exerts the opposite effect, indicating a causative role of BuChE downregulation in liver diseases. BuChE is predominantly expressed in hepatocytes in the liver, which can be reduced by TGFβ treatment. Notably, BuChE enzymatic activity is required to prevent HSC activation and fibrogenesis in homeostatic liver. As consequence of the lost BuChE metabolic activity, levels of released unmetabolized ACh are increased, which acts as DAMP to activate HSCs via ChRM3 signaling. This finding was confirmed using Chrm3ΔHSC mice, which exhibit diminished HSC activation and liver fibrosis. Conversely, ChRM3 overexpression in HSCs is promoting the fibrogenic process. Conclusion: Hepatocyte injury disrupts BuChE activity, causing local ACh accumulation and subsequent HSC activation via ChRM3 signaling. This novel cholinergic circuit drives fibrosis and represents a promising new target for therapeutic intervention.

ORGANISM(S): Mus musculus

PROVIDER: GSE325353 | GEO | 2026/03/24

REPOSITORIES: GEO

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Project description:Background & Aims: Butyrylcholinesterase (BuChE) is an enzyme involved in Alzheimer’s disease (AD), detoxification and depression. While BuChE expression is reduced in liver cirrhosis, its physiological substrate, acetylcholine (ACh), promotes fibrogenic responses in hepatic stellate cells (HSCs). However, the function of BuChE in liver fibrosis and mechanistic link between these two observations is not known. Methods: BuChE expression and HSC activation were assessed in liver biopsies from patients with liver fibrosis using qPCR and immunostaining. To investigate the functional role of BuChE in fibrogenesis, we employed BucheΔHep, knock-in (BucheKI/KI-Hep) mice, as well as pharmacological inhibition. The inhibitory effect of BuChE on HSC activation was further confirmed using conditioned medium from cells expressing mutant BuChE, in hepatocyte-HSC co-culture experiments, and analyses of recombinant BuChE protein. The release of ACh was measured with patch-clamp and the expression and functionality of its receptor were detected via intracellular Ca2+ influx measurements. The role of the muscarinic acetylcholine receptor 3 (ChRM3) in HSCs was investigated through gain- and loss-of-function experiments, utilizing Chrm3ΔHSC mice and AAV-mediated Chrm3 overexpression driven by the alpha-smooth muscle actin (Acta2) promoter. Results: Buche mRNA expression in livers and enzymatic activity in serum are downregulated in patients and mouse models with liver fibrosis, and show a negative correlation with F stage and ACTA2 expression. Pharmacological inhibition or depletion of BuChE in hepatocytes results in increased HSC activation and aggravates liver fibrosis in various mouse models, whereas its overexpression exerts the opposite effect, indicating a causative role of BuChE downregulation in liver diseases. BuChE is predominantly expressed in hepatocytes in the liver, which can be reduced by TGFβ treatment. Notably, BuChE enzymatic activity is required to prevent HSC activation and fibrogenesis in homeostatic liver. As consequence of the lost BuChE metabolic activity, levels of released unmetabolized ACh are increased, which acts as DAMP to activate HSCs via ChRM3 signaling. This finding was confirmed using Chrm3ΔHSC mice, which exhibit diminished HSC activation and liver fibrosis. Conversely, ChRM3 overexpression in HSCs is promoting the fibrogenic process. Conclusion: Hepatocyte injury disrupts BuChE activity, causing local ACh accumulation and subsequent HSC activation via ChRM3 signaling. This novel cholinergic circuit drives fibrosis and represents a promising new target for therapeutic intervention.

Project description:BACKGROUND AND AIM: We previously established that endoplasmic reticulum stress leads to unfolded protein response (UPR) that promotes liver fibrosis by activating hepatic stellate cell (HSC). We aimed to determine the role of X-box binding protein 1 (XBP1), one of three UPR effector pathways, in the fibrogenic HSC activation. METHODS: XBP1 expression was measured by qPCR in tunicamycin-treated human HSC lines (TWNT4 and LX2) and culture-activated human primary HSCs. XBP1 was overexpressed in primary human HSCs and the HSC lines, and fibrogenic genes (COL1A1, ACTA2, PDGFRB, MMP2, TIMP1) and encoded proteins were assessed by qPCR and Western blotting, respectively. Autophagy was inhibited by knockdown of ATG7. Genome-wide transcriptome profiling was performed to determine modulated molecular pathway by XBP1. In vivo XBP1 activation was assessed in transcriptome datasets of fibrotic mouse models and immunohistochemistry of human liver tissues. RESULTS: XBP1 overexpression accompanied both tunicamycin- and culture-based HSC activation. Ectopic overexpression of XBP1 induced fibrogenic gene expression in the HSC lines, which was inhibited by knockdown of ATG7. UPR pathway together with PDGFRB pathway, a hallmark of HSC activation, were induced in transcriptome profiles of XBP1-transduced HSC lines. Some known fibrogenic pathways such as transforming growth factor (TGF)-beta pathway were not induced, suggesting partial contribution of XBP1 to the entire fibrogenic HSC activation machinery. XBP1 target gene signature defined in our XBP1-overexpressing HSC lines was significantly induced in liver tissue transcriptome profiles of carbon tetrachloride-treated or bile duct-ligated mouse models, and XBP1 and alpha-smooth muscle actin (encoded by ACTA2, another hallmark of HSC activation) were co-localized in human fibrotic liver tissues, collectively supporting involvement of XBP1 in HSC activation and fibrogesis in vivo. CONCLUSIONS: XBP1-mediated UPR is at least partially responsible for fibrogenic HSC activation via autophagy.

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Butyrylcholinesterase alleviates hepatic stellate cell activation via paracrine muscarinic signaling [2W-CCL4-DOX-HSC]

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