Liver and Bile
J Hepatol. 2025;82(1):120-133
Targeting the liver clock improves fibrosis by restoring TGF-β signaling
Background and aims: Liver fibrosis is the major driver of hepatocellular carcinoma and liver disease-related death. Approved antifibrotic therapies are absent and compounds in development have limited efficacy. Increased TGF-β signaling drives collagen deposition by hepatic stellate cells (HSCs)/myofibroblasts. Here, the authors aimed to dissect the role of the circadian clock (CC) in controlling TGF-β signaling and liver fibrosis.
Methods: Using 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, the authors investigated the relationship between CC and TGF-β signaling. They explored hepatocyte-myofibroblast communication through bioinformatic analyses of single-nuclei transcriptomes and performed validation in cell-based models. Using mouse models for MASH (metabolic dysfunction-associated steatohepatitis)-related fibrosis and spheroids from patients with liver disease, they performed proof-of-concept studies to validate pharmacological targetability and clinical translatability.
Results: The authors discovered that the CC oscillator temporally gates TGF-β signaling and this regulation is broken in fibrosis. They 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 in patient-derived liver spheroids.
Conclusion: The circadian clock (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 have uncovered the CC as a novel therapeutic target for liver fibrosis – a growing unmet medical need.
DOI: 10.1016/j.jhep.2024.07.034
Prof. Dr. Dr. Bertram Bengsch
Section Head for Translational Systems Immunology in Hepatogastroenterology, Department of Internal Medicine II, University Medical Center Freiburg (Germany)
Tick, Tock liver clock: fibrosis as a consequence of TGF-β-driven disruption of hepatocyte circadian rhythms
Circadian rhythms are crucial for synchronizing physiological functions with day/night-cycle associated behavior both at systemic and cellular levels. As the central metabolic organ, the liver is tightly integrated into circadian rhythms that regulate digestive function. Disruption of circadian rhythms is known to drive metabolic liver disease. At the cellular level, transcriptional feedback loops involving core circadian clock (CC) genes (including PER, BMAL1, REV-ERBα and REV-ERBβ), also referred to as the CC oscillator system, are shared across many cell types and regulate rhythmic gene expression patterns together with tissue-specific transcription factors (DOI: 10.1055/a-1792-4240). In hepatocytes, the CC oscillator plays a prominent role in controlling metabolic gene expression. However, alterations in the hepatocyte clock also affect other interacting liver cell types (DOI: 10.1126/science.aba8984). Additionally, disruption of the hepatocyte clock promotes collagen deposition in metabolic dysfunction-associated steatotic liver disease (MASLD) (DOI: 10.3390/cells12121582). TGF-β signaling is a key driver of liver fibrosis and represents a promising therapeutic target (DOI: 10.1111/febs.13665). However, the mechanisms linking the liver clock to liver fibrosis and their connection to TGF-β signaling remain poorly understood.
In their study, Crouchet et al. tackled these unanswered questions by analyzing the role of the liver clock in both murine models of CC-mutant mice and in cell culture. They demonstrate that the liver CC regulates gene expression networks in hepatocytes that overlap with targets of TGF-β signaling. Moreover, disruption of the hepatocyte clock also influences hepatic stellate cells (HSCs), the primary pro-fibrotic cell type in the liver. In vitro experiments with primary human hepatocytes (PHHs) showed that free fatty acids (FFAs) – key drivers of metabolic dysfunction-associated steatohepatitis (MASH) pathogenesis – regulate CC-dependent gene expression and involve TGF-β target genes. Notably, REV-ERBα, a central component of the CC oscillator, is suppressed upon FFA exposure and in in vivo models of liver fibrosis. Treatment with a pharmacological REV-ERBα agonist inhibited TGF-β-dependent gene expression, while blocking pro-fibrotic TGF-β signaling in HSCs restored physiological CC gene expression. BMAL1 knockdown experiments in hepatocyte-HSC co-culture further revealed a role of the hepatocyte clock in regulating HSC gene expression, showing that the pro-fibrotic communication to HSCs is regulated by the CC. The authors then used sophisticated humanized liver chimeric mouse models (HLCMs), in which livers of RRG-NOD mice were repopulated with human PHHs. These mice were fed with a choline-deficient high fat diet (CD-HFD) to induce MASLD and fibrosis, and were treated with a REV-ERBα agonist. This treatment reduced liver fibrosis and also demonstrated antifibrotic effects in spheroids of patients with MASH or liver cirrhosis. Overall, the study highlights that MASH-associated pro-fibrotic intrahepatic cellular crosstalk between hepatocytes and HSCs involves TGF-β signaling regulating the CC. This mechanism is pharmacologically targetable: reactivation of the CC via REV-ERBα agonism may offer a novel therapeutic concept for treating liver fibrosis in patients with MASH. The study has other important implications, as additional strategies to alter CC activity, including behavior/diet-based and pharmacological interventions, may have synergistic effects.