p38γ is essential for cell cycle progression and liver tumorigenesis.

The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)-cyclin protein complex1. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show high expression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease.

The outcome of boosting mitochondrial activity in alcohol-associated liver disease is organ-dependent.

BACKGROUND AND AIMS: Alcohol-associated liver disease (ALD) accounts for 70% of liver-related deaths in Europe, with no effective approved therapies. Although mitochondrial dysfunction is one of the earliest manifestations of alcohol-induced injury, restoring mitochondrial activity remains a problematic strategy due to oxidative stress. Here, we identify methylation-controlled J protein (MCJ) as a mediator for ALD progression and hypothesize that targeting MCJ may help in recovering mitochondrial fitness without collateral oxidative damage. APPROACH AND RESULTS: C57BL/6 mice [wild-type (Wt)] Mcj knockout and Mcj liver-specific silencing (MCJ-LSS) underwent the NIAAA dietary protocol (Lieber-DeCarli diet containing 5% (vol/vol) ethanol for 10 days, plus a single binge ethanol feeding at day 11). To evaluate the impact of a restored mitochondrial activity in ALD, the liver, gut, and pancreas were characterized, focusing on lipid metabolism, glucose homeostasis, intestinal permeability, and microbiota composition. MCJ, a protein acting as an endogenous negative regulator of mitochondrial respiration, is downregulated in the early stages of ALD and increases with the severity of the disease. Whole-body deficiency of MCJ is detrimental during ALD because it exacerbates the systemic effects of alcohol abuse through altered intestinal permeability, increased endotoxemia, and dysregulation of pancreatic function, which overall worsens liver injury. On the other hand, liver-specific Mcj silencing prevents main ALD hallmarks, that is, mitochondrial dysfunction, steatosis, inflammation, and oxidative stress, as it restores the NAD + /NADH ratio and SIRT1 function, hence preventing de novo lipogenesis and improving lipid oxidation. CONCLUSIONS: Improving mitochondrial respiration by liver-specific Mcj silencing might become a novel therapeutic approach for treating ALD.

Hepatocyte dedifferentiation profiling in alcohol-related liver disease identifies CXCR4 as a driver of cell reprogramming.

BACKGROUND & AIMS: Loss of hepatocyte identity is associated with impaired liver function in alcohol-related hepatitis (AH). In this context, hepatocyte dedifferentiation gives rise to cells with a hepatobiliary (HB) phenotype expressing biliary and hepatocyte markers and showing immature features. However, the mechanisms and impact of hepatocyte dedifferentiation in liver disease are poorly understood. METHODS: HB cells and ductular reaction (DR) cells were quantified and microdissected from liver biopsies from patients with alcohol-related liver disease (ArLD). Hepatocyte-specific overexpression or deletion of C-X-C motif chemokine receptor 4 (CXCR4), and CXCR4 pharmacological inhibition were assessed in mouse liver injury. Patient-derived and mouse organoids were generated to assess plasticity. RESULTS: Here, we show that HB and DR cells are increased in patients with decompensated cirrhosis and AH, but only HB cells correlate with poor liver function and patients' outcome. Transcriptomic profiling of HB cells revealed the expression of biliary-specific genes and a mild reduction of hepatocyte metabolism. Functional analysis identified pathways involved in hepatocyte reprogramming, inflammation, stemness, and cancer gene programs. The CXCR4 pathway was highly enriched in HB cells and correlated with disease severity and hepatocyte dedifferentiation. In vitro, CXCR4 was associated with a biliary phenotype and loss of hepatocyte features. Liver overexpression of CXCR4 in chronic liver injury decreased the hepatocyte-specific gene expression profile and promoted liver injury. CXCR4 deletion or its pharmacological inhibition ameliorated hepatocyte dedifferentiation and reduced DR and fibrosis progression. CONCLUSIONS: This study shows the association of hepatocyte dedifferentiation with disease progression and poor outcome in AH. Moreover, the transcriptomic profiling of HB cells revealed CXCR4 as a new driver of hepatocyte-to-biliary reprogramming and as a potential therapeutic target to halt hepatocyte dedifferentiation in AH. IMPACT AND IMPLICATIONS: Here, we show that hepatocyte dedifferentiation is associated with disease severity and a reduced synthetic capacity of the liver. Moreover, we identify the CXCR4 pathway as a driver of hepatocyte dedifferentiation and as a therapeutic target in alcohol-related hepatitis. Therefore, this study reveals the importance of preserving strict control over hepatocyte plasticity in order to preserve liver function and promote tissue repair.

Novel therapeutic avenues for the study of chronic liver disease and regeneration: The foundation of the Iberoamerican Consortium for the study of liver Cirrhosis.

Unfortunately, there is a gap of understanding in the pathophysiology of chronic liver disease due to the lack of experimental models that exactly mimic the human disease. Additionally, the diagnosis of patients is very poor due to the lack of biomarkers than can detect the disease in early stages. Thus, it is of utmost interest the generation of a multidisciplinary consortium from different countries with a direct translation. The present reports the meeting of the 2021 Iberoamerican Consortium for the study of liver Cirrhosis, held online, in October 2021. The meeting, was focused on the recent advancements in the field of chronic liver disease and cirrhosis with a specific focus on cell pathobiology and liver regeneration, molecular and cellular targets involved in non-alcoholic hepatic steatohepatitis, alcoholic liver disease (ALD), both ALD and western diet, and end-stage liver cirrhosis and hepatocellular carcinoma. In addition, the meeting highlighted recent advances in targeted novel technology (-omics) and opening therapeutic avenues in this field of research.

Metabolic-associated fatty liver disease: From simple steatosis toward liver cirrhosis and potential complications. Proceedings of the Third Translational Hepatology Meeting, organized by the Spanish Association for the Study of the Liver (AEEH).

This is a meeting report of the 3rd Translational Hepatology Meeting held in Alicante, Spain, in October 2021. The meeting, which was organized by the Spanish Association for the Study of the Liver (AEEH), provided an update on the recent advances in the field of basic and translational hepatology, with a particular focus on the molecular and cellular mechanisms and therapeutic targets involved in metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH), cirrhosis and end-stage hepatocellular carcinoma (HCC).

Cyclin E1 and cyclin-dependent kinase 2 are critical for initiation, but not for progression of hepatocellular carcinoma.

E-type cyclins E1 (CcnE1) and E2 (CcnE2) are regulatory subunits of cyclin-dependent kinase 2 (Cdk2) and thought to control the transition of quiescent cells into the cell cycle. Initial findings indicated that CcnE1 and CcnE2 have largely overlapping functions for cancer development in several tumor entities including hepatocellular carcinoma (HCC). In the present study, we dissected the differential contributions of CcnE1, CcnE2, and Cdk2 for initiation and progression of HCC in mice and patients. To this end, we tested the HCC susceptibility in mice with constitutive deficiency for CcnE1 or CcnE2 as well as in mice lacking Cdk2 in hepatocytes. Genetic inactivation of CcnE1 largely prevented development of liver cancer in mice in two established HCC models, while ablation of CcnE2 had no effect on hepatocarcinogenesis. Importantly, CcnE1-driven HCC initiation was dependent on Cdk2. However, isolated primary hepatoma cells typically acquired independence on CcnE1 and Cdk2 with increasing progression in vitro, which was associated with a gene signature involving secondary induction of CcnE2 and up-regulation of cell cycle and DNA repair pathways. Importantly, a similar expression profile was also found in HCC patients with elevated CcnE2 expression and poor survival. In general, overall survival in HCC patients was synergistically affected by expression of CcnE1 and CcnE2, but not through Cdk2. Our study suggests that HCC initiation specifically depends on CcnE1 and Cdk2, while HCC progression requires expression of any E-cyclin, but no Cdk2.

Animal models for liver disease - A practical approach for translational research.

Animal models are crucial for improving our understanding of human pathogenesis, enabling researchers to identify therapeutic targets and test novel drugs. In the current review, we provide a comprehensive summary of the most widely used experimental models of chronic liver disease, starting from early stages of fatty liver disease (non-alcoholic and alcoholic) to steatohepatitis, advanced cirrhosis and end-stage primary liver cancer. We focus on aspects such as reproducibility and practicality, discussing the advantages and weaknesses of available models for researchers who are planning to perform animal studies in the near future. Additionally, we summarise current and prospective models based on human tissue bioengineering.

Pharmacological Inhibition of Cyclin-Dependent Kinases Triggers Anti-Fibrotic Effects in Hepatic Stellate Cells In Vitro.

Liver fibrosis is a wound healing process in response to chronic liver injury, which is characterized by the accumulation of extracellular collagen produced by Hepatic Stellate Cells (HSCs). This process involves cell cycle re-entry and proliferation of normally quiescent HSCs controlled by cyclins and associated cyclin-dependent kinases (Cdks). Cdk2 mediates the entry and progression through S-phase in complex with E-and A-type cyclins. We have demonstrated that cyclin E1 is essential for liver fibrogenesis in mice, but it is not known if this is dependent on Cdk2 or related Cdks. Here, we aimed to evaluate the benefit of the pan-Cdk inhibitor CR8 for treatment of liver fibrosis in vitro. CR8-treatment reduced proliferation and survival in immortalized HSC lines and in addition attenuated pro-fibrotic properties in primary murine HSCs. Importantly, primary murine hepatocytes were much more tolerant against the cytotoxic and anti-proliferative effects of CR8. We identified CR8 dosages mediating anti-fibrotic effects in primary HSCs without affecting cell cycle activity and survival in primary hepatocytes. In conclusion, the pharmacological pan-Cdk inhibitor CR8 restricts the pro-fibrotic properties of HSCs, while preserving proliferation and viability of hepatocytes at least in vitro. Therefore, CR8 and related drugs might be beneficial for the treatment of liver fibrosis.

Recent Advances in Practical Methods for Liver Cell Biology: A Short Overview.

Molecular and cellular research modalities for the study of liver pathologies have been tremendously improved over the recent decades. Advanced technologies offer novel opportunities to establish cell isolation techniques with excellent purity, paving the path for 2D and 3D microscopy and high-throughput assays (e.g., bulk or single-cell RNA sequencing). The use of stem cell and organoid research will help to decipher the pathophysiology of liver diseases and the interaction between various parenchymal and non-parenchymal liver cells. Furthermore, sophisticated animal models of liver disease allow for the in vivo assessment of fibrogenesis, portal hypertension and hepatocellular carcinoma (HCC) and for the preclinical testing of therapeutic strategies. The purpose of this review is to portray in detail novel in vitro and in vivo methods for the study of liver cell biology that had been presented at the workshop of the 8th meeting of the European Club for Liver Cell Biology (ECLCB-8) in October of 2018 in Bonn, Germany.

The Lieber-DeCarli Diet-A Flagship Model for Experimental Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is the most common chronic liver disease in the Western world, and it persists at a high prevalence. Understanding the pathophysiology and successful treatment for ALD is closely associated with the suitability of the animal model, which fully reflects all aspects of the pathogenesis and typical histological findings. This study reviews one of the most widely used experimental models of ALD in rodents-the Lieber-DeCarli (LDC) liquid diet. It is an easy, accurate, reliable, and inexpensive model to study the pathogenesis of early stages of ALD. Here, we discuss the historical background and provide an overview of the advantages and disadvantages of the classical LDC as well as modified "second-hit" models. We also provide a comprehensive protocol for the application of the LDC diet to perform it successfully, reliably, and reproducibly in mice.

Combined Activities of JNK1 and JNK2 in Hepatocytes Protect Against Toxic Liver Injury.

BACKGROUND & AIMS: c-Jun N-terminal kinase (JNK) 1 and JNK2 are expressed in hepatocytes and have overlapping and distinct functions. JNK proteins are activated via phosphorylation in response to acetaminophen- or carbon tetrachloride (CCl4)-induced liver damage; the level of activation correlates with the degree of injury. SP600125, a JNK inhibitor, has been reported to block acetaminophen-induced liver injury. We investigated the role of JNK in drug-induced liver injury (DILI) in liver tissue from patients and in mice with genetic deletion of JNK in hepatocytes. METHODS: We studied liver sections from patients with DILI (due to acetaminophen, phenprocoumon, nonsteroidal anti-inflammatory drugs, or autoimmune hepatitis) or patients without acute liver failure (controls) collected from a DILI Biobank in Germany. Levels of total and activated (phosphorylated) JNK were measured by immunohistochemistry and Western blotting. Mice with hepatocyte-specific deletion of Jnk1 (Jnk1(Δhepa)) or combination of Jnk1 and Jnk2 (Jnk(Δhepa)), as well as Jnk1-floxed C57BL/6 (control) mice, were given injections of CCl4 (to induce fibrosis) or acetaminophen (to induce toxic liver injury). We performed gene expression microarray and phosphoproteomic analyses to determine mechanisms of JNK activity in hepatocytes. RESULTS: Liver samples from DILI patients contained more activated JNK, predominantly in nuclei of hepatocytes and in immune cells, than healthy tissue. Administration of acetaminophen to Jnk(Δhepa) mice produced a greater level of liver injury than that observed in Jnk1(Δhepa) or control mice, based on levels of serum markers and microscopic and histologic analysis of liver tissues. Administration of CCl4 also induced stronger hepatic injury in Jnk(Δhepa) mice, based on increased inflammation, cell proliferation, and fibrosis progression, compared with Jnk1(Δhepa) or control mice. Hepatocytes from Jnk(Δhepa) mice given acetaminophen had an increased oxidative stress response, leading to decreased activation of adenosine monophosphate-activated protein kinase, total protein adenosine monophosphate-activated protein kinase levels, and pJunD and subsequent necrosis. Administration of SP600125 before or with acetaminophen protected Jnk(Δhepa) and control mice from liver injury. CONCLUSIONS: In hepatocytes, JNK1 and JNK2 appear to have combined effects in protecting mice from CCl4- and acetaminophen-induced liver injury. It is important to study the tissue-specific functions of both proteins, rather than just JNK1, in the onset of toxic liver injury. JNK inhibition with SP600125 shows off-target effects.

Bone marrow-derived c-jun N-terminal kinase-1 (JNK1) mediates liver regeneration.

Liver regeneration is controlled by a complex network of signaling molecules, and a prominent role for c-jun N-terminal kinase has been suggested during this process. In the present study, we aimed to characterize and define the cell-type-specific contribution of JNK1 activation during liver regeneration. We used hepatocyte-specific JNK1 knockout mice (JNK1(Δhepa)) using the cre/lox-P system. We performed partial hepatectomy (PH) in WT, JNK1(Δhepa) and JNK1(-/-) animals and investigated time-points up to 72 h after PH. Additionally, bone marrow transplantation experiments were conducted in order to identify the contribution of hematopoietic cell-derived JNK1 activation for liver regeneration. Our results show that liver regeneration was significantly impaired in JNK1(-/-) compared to JNK1(Δhepa) and WT animals. These data were evidenced by lower BrdU incorporation and decreased cell cycle markers such as Cyclin A, Cyclin D, E2F1 and PCNA 48 h after PH in JNK1(-/-) compared with JNK1(Δhepa) and WT livers. In JNK1(-/-) mice, our findings were associated with a reduced acute phase response as evidenced by a lower activation of the IL-6/STAT3/SAA-1 cascade. Additionally, CD11b(+)Ly6G(+)-cells were decreased in JNK1(-/-) compared with JNK1(Δhepa) and WT animals after PH. The transplantation of bone marrow-derived JNK1(-/-) into WT recipients caused significant reduction in liver regeneration. Interestingly, the transplantation of JNK1(-/-) into mice lacking JNK1 in hepatocytes only partially delayed liver regeneration. In summary, we provide evidence that (1) JNK1 in hematopoietic cells is crucial for liver regeneration, and (2) a synergistic function between JNK1 in hepatocytes and hematopoietic-derived cells is involved in the hepatic regenerative response.

Enhanced expression of c-myc in hepatocytes promotes initiation and progression of alcoholic liver disease.

BACKGROUND & AIMS: Progression of alcoholic liver disease (ALD) can be influenced by genetic factors, which potentially include specific oncogenes and tumor suppressors. In the present study, we tested the hypothesis that aberrant expression of the proto-oncogene c-myc might exert a crucial role in the development of ALD. METHODS: Expression of c-myc was measured in biopsies of patients with ALD by quantitative real-time PCR and immunohistochemistry. Mice with transgenic expression of c-myc in hepatocytes (alb-myc(tg)) and wild-type (WT) controls were fed either control or ethanol (EtOH) containing Lieber-DeCarli diet for 4weeks to induce ALD. RESULTS: Hepatic c-myc was strongly upregulated in human patients with advanced ALD and in EtOH-fed WT mice. Transcriptome analysis indicated deregulation of pathways involved in ER-stress, p53 signaling, hepatic fibrosis, cell cycle regulation, ribosomal synthesis and glucose homeostasis in EtOH-fed alb-myc(tg) mice. Transgenic expression of c-myc in hepatocytes with simultaneous EtOH-uptake led to early ballooning degeneration, increased liver collagen deposition and hepatic lipotoxicity, together with excessive CYP2E1-derived reactive oxygen species (ROS) production. Moreover, EtOH-fed alb-myc(tg) mice exhibited substantial changes in mitochondrial morphology associated with energy dysfunction. Pathway analysis revealed that elevated c-myc expression and ethanol uptake synergistically lead to strong AKT activation, Mdm2 phosphorylation and as a consequence to inhibition of p53. CONCLUSIONS: Expression of c-myc and EtOH-uptake synergistically accelerate the progression of ALD most likely due to loss of p53-dependent protection. Thus, c-myc is a new potential marker for the early detection of ALD and identification of risk patients.

The anti-fibrotic effects of CCN1/CYR61 in primary portal myofibroblasts are mediated through induction of reactive oxygen species resulting in cellular senescence, apoptosis and attenuated TGF-ß signaling.

UNLABELLED: Cysteine-rich protein 61 (CCN1/CYR61) is a CCN (CYR61, CTGF (connective tissue growth factor), and NOV (Nephroblastoma overexpressed gene)) family matricellular protein comprising six secreted CCN proteins in mammals. CCN1/CYR61 expression is associated with inflammation and injury repair. Recent studies show that CCN1/CYR61 limits fibrosis in models of cutaneous wound healing by inducing cellular senescence in myofibroblasts of the granulation tissue which thereby transforms into an extracellular matrix-degrading phenotype. We here investigate CCN1/CYR61 expression in primary profibrogenic liver cells (i.e., hepatic stellate cells and periportal myofibroblasts) and found an increase of CCN1/CYR61 expression during early activation of hepatic stellate cells that declines in fully transdifferentiated myofibroblasts. By contrast, CCN1/CYR61 levels found in primary parenchymal liver cells (i.e., hepatocytes) were relatively low compared to the levels exhibited in hepatic stellate cells and portal myofibroblasts. In models of ongoing liver fibrogenesis, elevated levels of CCN1/CYR61 were particularly noticed during early periods of insult, while expression declined during prolonged phases of fibrogenesis. We generated an adenovirus type 5 encoding CCN1/CYR61 (i.e., Ad5-CMV-CCN1/CYR61) and overexpressed CCN1/CYR61 in primary portal myofibroblasts. Interestingly, overexpressed CCN1/CYR61 significantly inhibited production of collagen type I at both mRNA and protein levels as evidenced by quantitative real-time polymerase chain reaction, Western blot and immunocytochemistry. CCN1/CYR61 further induces production of reactive oxygen species (ROS) leading to dose-dependent cellular senescence and apoptosis. Additionally, we demonstrate that CCN1/CYR61 attenuates TGF-ß signaling by scavenging TGF-ß thereby mitigating in vivo liver fibrogenesis in a bile duct ligation model. CONCLUSION: In line with dermal fibrosis and scar formation, CCN1/CYR61 is involved in liver injury repair and tissue remodeling. CCN1/CYR61 gene transfer into extracellular matrix-producing liver cells is therefore potentially beneficial in liver fibrotic therapy.

Haematopoietic cell-derived Jnk1 is crucial for chronic inflammation and carcinogenesis in an experimental model of liver injury.

BACKGROUND & AIMS: Chronic liver injury triggers complications such as liver fibrosis and hepatocellular carcinoma (HCC), which are associated with alterations in distinct signalling pathways. Of particular interest is the interaction between mechanisms controlled by IKKγ/NEMO, the regulatory IKK subunit, and Jnk activation for directing cell death and survival. In the present study, we aimed to define the relevance of Jnk in hepatocyte-specific NEMO knockout mice (NEMO(Δhepa)), a genetic model of chronic inflammatory liver injury. METHODS: We generated Jnk1(-/-)/NEMO(Δhepa) and Jnk2(-/-)/NEMO(Δhepa) mice by crossing NEMO(Δhepa) mice with Jnk1 and Jnk2 global deficient animals, respectively, and examined the progression of chronic liver disease. Moreover, we investigated the expression of Jnk during acute liver injury, evaluated the role of Jnk1 in bone marrow-derived cells, and analysed the expression of NEMO and p-JNK in human diseased-livers. RESULTS: Deletion of Jnk1 significantly aggravated the progression of liver disease, exacerbating apoptosis, compensatory proliferation and carcinogenesis in NEMO(Δhepa) mice. Conversely, Jnk2(-/-)/NEMO(Δhepa) displayed hepatic inflammation. By using bone marrow transfer, we observed that Jnk1 in haematopoietic cells had an impact on the progression of chronic liver disease in NEMO(Δhepa) livers. These findings are of clinical relevance since NEMO expression was downregulated in hepatocytes of patients with HCC whereas NEMO and p-JNK were expressed in a large amount of infiltrating cells. CONCLUSIONS: A synergistic function of Jnk1 in haematopoietic cells and hepatocytes might be relevant for the development of chronic liver injury. These results elucidate the complex function of Jnk in chronic inflammatory liver disease.

Concurrent deletion of cyclin E1 and cyclin-dependent kinase 2 in hepatocytes inhibits DNA replication and liver regeneration in mice.

UNLABELLED: The liver has a strong regenerative capacity. After injury, quiescent hepatocytes can reenter the mitotic cell cycle to restore tissue homeostasis. This G(0) /G(1) -S cell-cycle transition of primed hepatocytes is regulated by complexes of cyclin-dependent kinase 2 (Cdk2) with E-type cyclins (CcnE1 or CcnE2). However, single genetic ablation of either E-cyclin or Cdk2 does not affect overall liver regeneration. Here, we systematically investigated the contribution of CcnE1, CcnE2, and Cdk2 for liver regeneration after partial hepatectomy (PH) by generating corresponding double- and triple-knockout (KO) mouse mutants. We demonstrate that conditional deletion of Cdk2 alone in hepatocytes resulted in accelerated induction of CcnE1, but otherwise normal initiation of S phase in vivo and in vitro. Excessive CcnE1 did not contribute to a noncanonical kinase activity, but was located at chromatin together with components of the pre-replication complex (pre-RC), such as the minichromosome maintenance (MCM) helicase. Concomitant ablation of Cdk2 and CcnE1 in hepatocytes caused a defect in pre-RC formation and further led to dramatically impaired S-phase progression by down-regulation of cyclin A2 and cell death in vitro and substantially reduced hepatocyte proliferation and liver regeneration after PH in vivo. Similarly, combined loss of CcnE1 and CcnE2, but also the Cdk2/CcnE1/CcnE2 triple KO in liver, significantly inhibited S-phase initiation and liver mass reconstitution after PH, whereas concomitant ablation of CcnE2 and Cdk2 had no effect. CONCLUSION: In the absence of Cdk2, CcnE1 performs crucial kinase-independent functions in hepatocytes, which are capable of driving MCM loading on chromatin, cyclin A2 expression, and S-phase progression. Thus, combined inactivation of Cdk2 and CcnE1 is the minimal requirement for blocking S-phase machinery in vivo.

Jnk1 in murine hepatic stellate cells is a crucial mediator of liver fibrogenesis.

OBJECTIVE: The c-Jun N-terminal kinase-1 (Jnk1) gene has been shown to be involved in liver fibrosis. Here, we aimed to investigate the molecular mechanism and define the cell type involved in mediating the Jnk1-dependent effect on liver fibrogenesis. DESIGN: Jnk1(f/f) wildtype (WT), Jnk1(-/-) and Jnk1(Δhepa) (hepatocyte-specific deletion of Jnk1) mice were subjected to (i) bile duct ligation (BDL) and (ii) CCl4-induced liver fibrosis. Additionally, we performed bone marrow transplantations (BMT), isolated primary hepatic stellate cells (HSCs), studied their activation in vitro and investigated human diseased liver samples. RESULTS: Phosphorylated Jnk was expressed in myofibroblasts, epithelial and inflammatory cells during the progression of fibrogenesis in humans and mice. In mice, liver transaminases, alkaline phosphatase, bilirubin and liver histology revealed reduced injury in Jnk1(-/-) compared with WT and Jnk1(Δhepa) mice correlating with lower hepatocyte cell death and proliferation. Consequently, parameters of liver fibrosis such as Sirius red staining and collagen IA1 and α-smooth muscle actin expression were downregulated in Jnk1(-/-) compared with WT and Jnk1(Δhepa) livers, 4 weeks after CCl4 or BDL. BMT experiments excluded bone marrow-derived cells from having a major impact on the Jnk1-dependent effect on fibrogenesis, while primary HSCs from Jnk1(-/-) livers showed reduced transdifferentiation and extracellular matrix production. Moreover, Jnk1 ablation caused a reduced lifespan and poor differentiation of HSCs into matrix-producing myofibroblasts. CONCLUSIONS: Jnk1 in HSCs, but not in hepatocytes, significantly contribute to liver fibrosis development, identifying Jnk1 in HSCs as a profibrotic kinase and a promising cell-directed target for liver fibrosis.

Overexpression of c-myc in hepatocytes promotes activation of hepatic stellate cells and facilitates the onset of liver fibrosis.

BACKGROUND: Liver fibrosis is a consequence of chronic liver injury and can further progress to hepatocellular carcinoma (HCC). Fibrogenesis involves activation of hepatic stellate cells (HSC) and proliferation of hepatocytes upon liver injury. HCC is frequently associated with overexpression of the proto-oncogene c-myc. However, the impact of c-myc for initiating pathological precursor stages such as liver fibrosis is poorly characterized. In the present study we thus investigated the impact of c-myc for liver fibrogenesis. METHODS: Expression of c-myc was measured in biopsies of patients with liver fibrosis of different etiologies by quantitative real-time PCR (qPCR). Primary HSC were isolated from mice with transgenic overexpression of c-myc in hepatocytes (alb-myc(tg)) and wildtype (WT) controls and investigated for markers of cell cycle progression and fibrosis by qPCR and immunofluorescence microscopy. Liver fibrosis in WT and alb-myc(tg) mice was induced by repetitive CCl4 treatment. RESULTS: We detected strong up-regulation of hepatic c-myc in patients with advanced liver fibrosis. In return, overexpression of c-myc in alb-myc(tg) mice resulted in increased liver collagen deposition and induction of α-smooth-muscle-actin indicating HSC activation. Primary HSC derived from alb-myc(tg) mice showed enhanced proliferation and accelerated transdifferentiation into myofibroblasts in vitro. Accordingly, fibrosis initiation in vivo after chronic CCl4 treatment was accelerated in alb-myc(tg) mice compared to controls. CONCLUSION: Overexpression of c-myc is a novel marker of liver fibrosis in man and mice. We conclude that chronic induction of c-myc especially in hepatocytes has the potential to prime resident HSC for activation, proliferation and myofibroblast differentiation.

Seladelpar: New hope for patients with primary biliary cholangitis.

The study by Hirschfield et al.1 demonstrated safety profile and clinically significant effectiveness of the peroxisome proliferator-activated receptor delta (PPARδ) agonist seladelpar in patients with primary biliary cholangitis, highlighting its plausible use as a second-line treatment to reduce disease activity and pruritus.

Breaking the barriers: the role of gut homeostasis in Metabolic-Associated Steatotic Liver Disease (MASLD).

Obesity, insulin resistance (IR), and the gut microbiome intricately interplay in Metabolic-associated Steatotic Liver Disease (MASLD), previously known as Non-Alcoholic Fatty Liver Disease (NAFLD), a growing health concern. The complex progression of MASLD extends beyond the liver, driven by "gut-liver axis," where diet, genetics, and gut-liver interactions influence disease development. The pathophysiology of MASLD involves excessive liver fat accumulation, hepatocyte dysfunction, inflammation, and fibrosis, with subsequent risk of hepatocellular carcinoma (HCC). The gut, a tripartite barrier, with mechanical, immune, and microbial components, engages in a constant communication with the liver. Recent evidence links dysbiosis and disrupted barriers to systemic inflammation and disease progression. Toll-like receptors (TLRs) mediate immunological crosstalk between the gut and liver, recognizing microbial structures and triggering immune responses. The "multiple hit model" of MASLD development involves factors like fat accumulation, insulin resistance, gut dysbiosis, and genetics/environmental elements disrupting the gut-liver axis, leading to impaired intestinal barrier function and increased gut permeability. Clinical management strategies encompass dietary interventions, physical exercise, pharmacotherapy targeting bile acid (BA) metabolism, and microbiome modulation approaches through prebiotics, probiotics, symbiotics, and fecal microbiota transplantation (FMT). This review underscores the complex interactions between diet, metabolism, microbiome, and their impact on MASLD pathophysiology and therapeutic prospects.