Targeting the liver clock improves fibrosis by restoring TGF-β signaling

Highlights•The circadian clock (CC) is a regulator of TGF-β signaling in the healthy liver.•The breakdown of CC control in MASH is associated with liver fibrosis progression.•Proof-of-concept studies in several models unravel targetability by a small molecule.•Pharmacological targeting the CC provides opportunities to improve liver fibrosis.AbstractBackground & AimsLiver fibrosis is the major driver for hepatocellular carcinoma and liver disease related death. Approved anti-fibrotic therapies are absent and compounds in development have limited efficacy. Increased TGF-β signaling drives collagen deposition by hepatic stellate cells (HSC)/myofibroblasts. Here, we aimed to dissect the role of the circadian clock (CC) in controlling TGF-β signaling and liver fibrosis.MethodsUsing CC-mutant mice, enriched HSCs and myofibroblasts obtained from healthy and fibrotic mice in different CC-phases and loss-of-function studies in human hepatocytes and myofibroblasts, we investigated the relationship between CC and TGF-β signaling. We explored hepatocyte-myofibroblast communication through bioinformatic analyses of single-nuclei transcriptomes and validation in cell-based models. Using mouse models for MASH fibrosis and spheroids from patients with liver disease, we performed proof-of-concept studies to validate pharmacological targetability and clinical translatability.ResultsWe discovered that the CC-oscillator temporally gates TGF-β signaling and this regulation is broken in fibrosis. We demonstrate that HSCs and myofibroblasts contain a functional CC with rhythmic expression of numerous genes, including fibrogenic genes. Perturbation studies in hepatocytes and myofibroblasts revealed a reciprocal relationship between TGF-β-activation and CC perturbation, which was confirmed in patient-derived ex vivo and in vivo models. Pharmacological modulation of CC-TGF-β signaling inhibited fibrosis in mouse models in vivo as well as patient-derived liver spheroids.ConclusionThe CC regulates TGF-β signaling, and the breakdown of this control is associated with liver fibrosis in patients. Pharmacological proof-of-concept studies across different models uncover the CC as a therapeutic candidate target for liver fibrosis – a rising global unmet medical need.Impact and implicationsLiver fibrosis due to metabolic diseases is a global health challenge. Many liver functions are rhythmic throughout the day being controlled by the circadian clock (CC). Here we demonstrate that the regulation of the CC is perturbed upon chronic liver injury and this perturbation contributes to fibrotic disease. By showing that a compound targeting the CC improves liver fibrosis in patient-derived models, this study provides a novel therapeutic candidate strategy to treat fibrosis in patients. Additional studies will be needed for clinical translation. Since the findings uncovers a previously undiscovered profibrotic mechanism and therapeutic target, the study is of interest for scientists investigating liver disease, clinical hepatologists and drug developers.Graphical abstractRegulation of TGFβ expression by the liver circadian clock in the healthy and fibrotic liver. In the healthy liver, the CC-oscillator controls the diurnal expression of numerous genes including TGFβ-SMAD pathway members. In MASH-induced liver fibrosis, the perturbation of the CC-oscillator leads to dysregulation of the TGFβ-SMAD pathway and perturbed hepatocyte and myofibroblast communication. Pharmacological targeting of the CC by an REV-ERBα agonist reduces TGF-β activity and restores cell-cell communication with inhibition of fibrosis progression.Footnote: abbreviation graphical abstract: CC: circadian clock; HSC: hepatic stellate cells; HLMF: human liver myofibroblast; MASH: metabolic dysfunction-associated steatohepatitis; TGFβ: transforming growth factor beta.