The hepatocyte epidermal growth factor receptor (EGFR) pathway regulates the cellular interactome within the liver fibrotic niche.

Liver fibrosis is the consequence of chronic liver injury in the presence of an inflammatory component. Although the main executors of this activation are known, the mechanisms that lead to the inflammatory process that mediates the production of pro-fibrotic factors are not well characterized. Epidermal growth factor receptor (EGFR) signaling in hepatocytes is essential for the regenerative processes of the liver; however, its potential role in regulating the fibrotic niche is not yet clear. Our group generated a mouse model that expresses an inactive truncated form of the EGFR specifically in hepatocytes (ΔEGFR mice). Here, we have analyzed the response of WT and ΔEGFR mice to chronic treatment with carbon tetrachloride (CCl4), which induces a pro-inflammatory and fibrotic process in the liver. The results indicated that the hallmarks of liver fibrosis were attenuated in CCl4-treated ΔEGFR mice when compared with CCl4-treated WT mice, coinciding with a faster resolution of the fibrotic process and ameliorated damage. The absence of EGFR activity in hepatocytes induced changes in the pattern of immune cells in the liver, with a notable increase in the population of M2 macrophages, more related to fibrosis resolution, as well as in the population of lymphocytes related to eradication of the damage. Transcriptome analysis of hepatocytes, and secretome studies of extracellular media from in vitro experiments, allowed us to elucidate the specific molecular mechanisms regulated by EGFR that mediate hepatocyte production of both pro-fibrotic and pro-inflammatory mediators; these have consequences for the deposition of extracellular matrix proteins, as well as for the immune microenvironment. Overall, our study uncovered novel mechanistic insights regarding EGFR kinase-dependent actions in hepatocytes that reveal its key role in chronic liver damage. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Toward the use of novel alternative methods in epilepsy modeling and drug discovery.

Epilepsy is a chronic brain disease and, considering the amount of people affected of all ages worldwide, one of the most common neurological disorders. Over 20 novel antiseizure medications (ASMs) have been released since 1993, yet despite substantial advancements in our understanding of the molecular mechanisms behind epileptogenesis, over one-third of patients continue to be resistant to available therapies. This is partially explained by the fact that the majority of existing medicines only address seizure suppression rather than underlying processes. Understanding the origin of this neurological illness requires conducting human neurological and genetic studies. However, the limitation of sample sizes, ethical concerns, and the requirement for appropriate controls (many patients have already had anti-epileptic medication exposure) in human clinical trials underscore the requirement for supplemental models. So far, mammalian models of epilepsy have helped to shed light on the underlying causes of the condition, but the high costs related to breeding of the animals, low throughput, and regulatory restrictions on their research limit their usefulness in drug screening. Here, we present an overview of the state of art in epilepsy modeling describing gold standard animal models used up to date and review the possible alternatives for this research field. Our focus will be mainly on ex vivo, in vitro, and in vivo larval zebrafish models contributing to the 3R in epilepsy modeling and drug screening. We provide a description of pharmacological and genetic methods currently available but also on the possibilities offered by the continued development in gene editing methodologies, especially CRISPR/Cas9-based, for high-throughput disease modeling and anti-epileptic drugs testing.

The NADPH oxidase NOX4 regulates redox and metabolic homeostasis preventing HCC progression.

BACKGROUND AND AIMS: The NADPH oxidase NOX4 plays a tumor-suppressor function in HCC. Silencing NOX4 confers higher proliferative and migratory capacity to HCC cells and increases their in vivo tumorigenic potential in xenografts in mice. NOX4 gene deletions are frequent in HCC, correlating with higher tumor grade and worse recurrence-free and overall survival rates. However, despite the accumulating evidence of a protective regulatory role in HCC, the cellular processes governed by NOX4 are not yet understood. Accordingly, the aim of this work was to better understand the molecular mechanisms regulated by NOX4 in HCC in order to explain its tumor-suppressor action. APPROACH AND RESULTS: Experimental models: cell-based loss or gain of NOX4 function experiments, in vivo hepatocarcinogenesis induced by diethylnitrosamine in Nox4 -deficient mice, and analyses in human HCC samples. Methods include cellular and molecular biology analyses, proteomics, transcriptomics, and metabolomics, as well as histological and immunohistochemical analyses in tissues. Results identified MYC as being negatively regulated by NOX4. MYC mediated mitochondrial dynamics and a transcriptional program leading to increased oxidative metabolism, enhanced use of both glucose and fatty acids, and an overall higher energetic capacity and ATP level. NOX4 deletion induced a redox imbalance that augmented nuclear factor erythroid 2-related factor 2 (Nrf2) activity and was responsible for MYC up-regulation. CONCLUSIONS: Loss of NOX4 in HCC tumor cells induces metabolic reprogramming in a Nrf2/MYC-dependent manner to promote HCC progression.

Mitochondrial bioenergetics boost macrophage activation, promoting liver regeneration in metabolically compromised animals.

BACKGROUND AND AIMS: Hepatic ischemia-reperfusion injury (IRI) is the leading cause of early posttransplantation organ failure as mitochondrial respiration and ATP production are affected. A shortage of donors has extended liver donor criteria, including aged or steatotic livers, which are more susceptible to IRI. Given the lack of an effective treatment and the extensive transplantation waitlist, we aimed at characterizing the effects of an accelerated mitochondrial activity by silencing methylation-controlled J protein (MCJ) in three preclinical models of IRI and liver regeneration, focusing on metabolically compromised animal models. APPROACH AND RESULTS: Wild-type (WT), MCJ knockout (KO), and Mcj silenced WT mice were subjected to 70% partial hepatectomy (Phx), prolonged IRI, and 70% Phx with IRI. Old and young mice with metabolic syndrome were also subjected to these procedures. Expression of MCJ, an endogenous negative regulator of mitochondrial respiration, increases in preclinical models of Phx with or without vascular occlusion and in donor livers. Mice lacking MCJ initiate liver regeneration 12 h faster than WT and show reduced ischemic injury and increased survival. MCJ knockdown enables a mitochondrial adaptation that restores the bioenergetic supply for enhanced regeneration and prevents cell death after IRI. Mechanistically, increased ATP secretion facilitates the early activation of Kupffer cells and production of TNF, IL-6, and heparin-binding EGF, accelerating the priming phase and the progression through G1 /S transition during liver regeneration. Therapeutic silencing of MCJ in 15-month-old mice and in mice fed a high-fat/high-fructose diet for 12 weeks improves mitochondrial respiration, reduces steatosis, and overcomes regenerative limitations. CONCLUSIONS: Boosting mitochondrial activity by silencing MCJ could pave the way for a protective approach after major liver resection or IRI, especially in metabolically compromised, IRI-susceptible organs.

Clathrin switches transforming growth factor-ß role to pro-tumorigenic in liver cancer.

BACKGROUND & AIMS: Upon ligand binding, tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR), are recruited into clathrin-coated pits for internalization by endocytosis, which is relevant for signalling and/or receptor degradation. In liver cells, transforming growth factor-ß (TGF-ß) induces both pro- and anti-apoptotic signals; the latter are mediated by the EGFR pathway. Since EGFR mainly traffics via clathrin-coated vesicles, we aimed to analyse the potential role of clathrin in TGF-ß-induced signalling in liver cells and its relevance in liver cancer. METHODS: Real-Time PCR and immunohistochemistry were used to analyse clathrin heavy-chain expression in human (CLTC) and mice (Cltc) liver tumours. Transient knockdown (siRNA) or overexpression of CLTC were used to analyse its role on TGF-ß and EGFR signalling in vitro. Bioinformatic analysis was used to determine the effect of CLTC and TGFB1 expression on prognosis and overall survival in patients with hepatocellular carcinoma (HCC). RESULTS: Clathrin expression increased during liver tumorigenesis in humans and mice. CLTC knockdown cells responded to TGF-ß phosphorylating SMADs (canonical signalling) but showed impairment in the anti-apoptotic signals (EGFR transactivation). Experiments of loss or gain of function in HCC cells reveal an essential role for clathrin in inhibiting TGF-ß-induced apoptosis and upregulation of its pro-apoptotic target NOX4. Autocrine TGF-ß signalling in invasive HCC cells upregulates CLTC expression, switching its role to pro-tumorigenic. A positive correlation between TGFB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGFB1 and CLTC had a worse prognosis and lower overall survival. CONCLUSIONS: This work describes a novel role for clathrin in liver tumorigenesis, favouring non-canonical pro-tumorigenic TGF-ß pathways. CLTC expression in human HCC samples could help select patients that would benefit from TGF-ß-targeted therapy. LAY SUMMARY: Clathrin heavy-chain expression increases during liver tumorigenesis in humans (CLTC) and mice (Cltc), altering the cellular response to TGF-ß in favour of anti-apoptotic/pro-tumorigenic signals. A positive correlation between TGFB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGFB1 and CLTC had a worse prognosis and lower overall survival. CLTC expression in HCC human samples could help select patients that would benefit from therapies targeting TGF-ß.

Paradoxical role of the NADPH oxidase NOX4 in early preneoplastic stages of hepatocytes induced by amino acid deprivation.

BACKGROUND: The NADPH oxidase (NOX) 4 is an important source of ROS in signal transduction that acts as a liver tumor suppressor. Transforming Growth Factor ß (TGF-ß) and Epidermal Growth Factor Receptor (EGFR) pathways are involved in the modulation of NOX4 expression. Data showed that recurrent protein deprivation induces changes distinctive of a preneoplastic profile. However, the mechanisms underneath these changes have not been completely understood. METHODS: Hepatocytes that survived to the lack of amino acids (Aa) (Sel line) were cultured in complete or Aa free medium. We elucidated the molecular mechanisms that support such preneoplastic alterations employing biochemical and molecular biology assays. RESULTS: Sel line showed increased phospho-AKT and phospho-ERKs levels, diminished caspase-3 activity, augmented cell proliferation and overactivation of EGFR pathway, reminiscent of a preneoplastic phenotype. NOX4 was upregulated in these cells by TGF-ß canonical pathway, however ROS levels were not found increased as a result of an increment of antioxidant enzymes. Inhibition of TGF-ß receptor diminished NOX4 and strikingly, after EGFR inhibition, NOX4 levels also decreased. Therefore, both TGF-ß and EGFR pathways are shown to be involved in the upregulation of NOX4 in Sel line. CONCLUSIONS: This work provides novel results regarding to the regulation of NOX4 in the preneoplastic transformation of hepatocytes in the absence of Aa, in the context of TGF-ß and EGFR pathways. GENERAL SIGNIFICANCE: The advances in the understanding of the molecular mechanisms whose deregulation ultimately causes Hepatocellular Carcinoma (HCC) are essential to prevent it and to design diagnostic biomarkers and therapeutic tools.

Transforming Growth Factor-ß-Induced Cell Plasticity in Liver Fibrosis and Hepatocarcinogenesis.

The Transforming Growth Factor-beta (TGF-ß) family plays relevant roles in the regulation of different cellular processes that are essential for tissue and organ homeostasis. In the case of the liver, TGF-ß signaling participates in different stages of disease progression, from initial liver injury toward fibrosis, cirrhosis and cancer. When a chronic injury takes place, mobilization of lymphocytes and other inflammatory cells occur, thus setting the stage for persistence of an inflammatory response. Macrophages produce profibrotic mediators, among them, TGF-ß, which is responsible for activation -transdifferentiation- of quiescent hepatic stellate cells (HSC) to a myofibroblast (MFB) phenotype. MFBs are the principal source of extracellular matrix protein (ECM) accumulation and prominent mediators of fibrogenesis. TGF-ß also mediates an epithelial-mesenchymal transition (EMT) process in hepatocytes that may contribute, directly or indirectly, to increase the MFB population. In hepatocarcinogenesis, TGF-ß plays a dual role, behaving as a suppressor factor at early stages, but contributing to later tumor progression, once cells escape from its cytostatic effects. As part of its potential pro-tumorigenic actions, TGF-ß induces EMT in liver tumor cells, which increases its pro-migratory and invasive potential. In parallel, TGF-ß also induces changes in tumor cell plasticity, conferring properties of a migratory tumor initiating cell (TIC). The main aim of this review is to shed light about the pleiotropic actions of TGF-ß that explain its effects on the different liver cell populations. The cross-talk with other signaling pathways that contribute to TGF-ß effects, in particular the Epidermal Growth Factor Receptor (EGFR), will be presented. Finally, we will discuss the rationale for targeting the TGF-ß pathway in liver pathologies.

TGF-ß and the Tissue Microenvironment: Relevance in Fibrosis and Cancer.

Transforming growth factor-β (TGF-β) is a cytokine essential for the induction of the fibrotic response and for the activation of the cancer stroma. Strong evidence suggests that a strong cross-talk exists among TGF-β and the tissue extracellular matrix components. TGF-β is stored in the matrix as part of a large latent complex bound to the latent TGF-β binding protein (LTBP) and matrix binding of latent TGF-β complexes, which is required for an adequate TGF-β function. Once TGF-β is activated, it regulates extracellular matrix remodelling and promotes a fibroblast to myofibroblast transition, which is essential in fibrotic processes. This cytokine also acts on other cell types present in the fibrotic and tumour microenvironment, such as epithelial, endothelial cells or macrophages and it contributes to the cancer-associated fibroblast (CAF) phenotype. Furthermore, TGF-β exerts anti-tumour activity by inhibiting the host tumour immunosurveillance. Aim of this review is to update how TGF-β and the tissue microenvironment cooperate to promote the pleiotropic actions that regulate cell responses of different cell types, essential for the development of fibrosis and tumour progression. We discuss recent evidences suggesting the use of TGF-β chemical inhibitors as a new line of defence against fibrotic disorders or cancer.

The level of caveolin-1 expression determines response to TGF-ß as a tumour suppressor in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a heterogeneous tumour associated with poor prognostic outcome. Caveolin-1 (CAV1), a membrane protein involved in the formation of caveolae, is frequently overexpressed in HCC. Transforming growth factor-beta (TGF-ß) is a pleiotropic cytokine having a dual role in hepatocarcinogenesis: inducer of apoptosis at early phases, but pro-tumourigenic once cells acquire mechanisms to overcome its suppressor effects. Apoptosis induced by TGF-ß is mediated by upregulation of the NADPH oxidase NOX4, but counteracted by transactivation of the epidermal growth factor receptor (EGFR) pathway. Previous data suggested that CAV1 is required for the anti-apoptotic signals triggered by TGF-ß in hepatocytes. Whether this mechanism is relevant in hepatocarcinogenesis has not been explored yet. Here we analysed the TGF-ß response in HCC cell lines that express different levels of CAV1. Accordingly, stable CAV1 knockdown or overexpressing cell lines were generated. We demonstrate that CAV1 is protecting HCC cells from TGF-ß-induced apoptosis, which attenuates its suppressive effect on clonogenic growth and increases its effects on cell migration. Downregulation of CAV1 in HLE cells promotes TGF-ß-mediated induction of the pro-apoptotic BMF, which correlates with upregulation of NOX4, whereas CAV1 overexpression in Huh7 cells shows the opposite effect. CAV1 silenced HLE cells show attenuation in TGF-ß-induced EGFR transactivation and activation of the PI3K/AKT pathway. On the contrary, Huh7 cells, which do not respond to TGF-ß activating the EGFR pathway, acquire the capacity to do so when CAV1 is overexpressed. Analyses in samples from HCC patients revealed that tumour tissues presented higher expression levels of CAV1 compared with surrounding non-tumoural areas. Furthermore, a significant positive correlation among the expression of CAV1 and TGFB1 was observed. We conclude that CAV1 has an essential role in switching the response to TGF-ß from cytostatic to tumourigenic, which could have clinical meaning in patient stratification.

Dissecting the role of epidermal growth factor receptor catalytic activity during liver regeneration and hepatocarcinogenesis.

UNLABELLED: Different data support a role for the epidermal growth factor receptor (EGFR) pathway during liver regeneration and hepatocarcinogenesis. However, important issues, such as the precise mechanisms mediating its actions and the unique versus redundant functions, have not been fully defined. Here, we present a novel transgenic mouse model expressing a hepatocyte-specific truncated form of human EGFR, which acts as negative dominant mutant (ΔEGFR) and allows definition of its tyrosine kinase-dependent functions. Results indicate a critical role for EGFR catalytic activity during the early stages of liver regeneration. Thus, after two-thirds partial hepatectomy, ΔEGFR livers displayed lower and delayed proliferation and lower activation of proliferative signals, which correlated with overactivation of the transforming growth factor-ß pathway. Altered regenerative response was associated with amplification of cytostatic effects of transforming growth factor-ß through induction of cell cycle negative regulators. Interestingly, lipid synthesis was severely inhibited in ΔEGFR livers after partial hepatectomy, revealing a new function for EGFR kinase activity as a lipid metabolism regulator in regenerating hepatocytes. In spite of these profound alterations, ΔEGFR livers were able to recover liver mass by overactivating compensatory signals, such as c-Met. Our results also indicate that EGFR catalytic activity is critical in the early preneoplastic stages of the liver because ΔEGFR mice showed a delay in the appearance of diethyl-nitrosamine-induced tumors, which correlated with decreased proliferation and delay in the diethyl-nitrosamine-induced inflammatory process. CONCLUSION: These studies demonstrate that EGFR catalytic activity is critical during the initial phases of both liver regeneration and carcinogenesis and provide key mechanistic insights into how this kinase acts to regulate liver pathophysiology. (Hepatology 2016;63:604-619).