Revisiting the liver: from development to regeneration - what we ought to know!

The liver is structurally and functionally heterogeneous and complex, and it accomplishes crucial functions for the organism. Its most remarkable potential is its capacity to regenerate after injury in order to maintain whole body homeostasis and guarantee the survival of the individual. Under normal conditions, liver regeneration (LR) is attributed to adult hepatocytes, the main cells in the liver which are able to proliferate in response to different stimuli or injuries. Nevertheless, when liver injury is severe and/or hepatocytes are prevented from proliferation, liver stem/progenitor cells (LS/PCs) participate directing LR to maintain liver mass and functions. Different mechanisms have been shown to guide this second line of LR, such as intrahepatic and extrahepatic liver progenitor cells, as well as transdifferentiation processes between hepatocytes and other liver cells. For this reason, many efforts have been made to elucidate the specific molecular mechanisms which orchestrate this process; this in turn would improve the prognosis and treatment of liver diseases. In this review, we revisit the fascinating process of LR, also with a short overview about liver development, the process from which arises the concept of LS/PCs participating in LR, and very important nowadays when considering cell therapy and tissue bioengineering for the treatment of patients suffering from liver disease

Downregulation of epidermal growth factor receptor in hepatocellular carcinoma facilitates transforming growth factor-β-induced epithelial to amoeboid transition

The Epidermal Growth Factor Receptor (EGFR) and the Transforming Growth Factor-beta (TGF-β) are key regulators of hepatocarcinogenesis. Targeting EGFR was proposed as a promising therapy; however, poor success was obtained in human hepatocellular carcinoma (HCC) clinical trials. Here, we describe how EGFR is frequently downregulated in HCC patients while TGF-β is upregulated. Using 2D/3D cellular models, we show that after EGFR loss, TGF-β is more efficient in its pro-migratory and invasive effects, inducing epithelial to amoeboid transition. EGFR knock-down promotes loss of cell-cell and cell-to-matrix adhesion, favouring TGF-β-induced actomyosin contractility and acquisition of an amoeboid migratory phenotype. Moreover, TGF-β upregulates RHOC and CDC42 after EGFR silencing, promoting Myosin II in amoeboid cells. Importantly, low EGFR combined with high TGFB1 or RHOC/CDC42 levels confer poor patient prognosis. In conclusion, this work reveals a new tumour suppressor function for EGFR counteracting TGF-β-mediated epithelial to amoeboid transitions in HCC, supporting a rational for targeting the TGF-β pathway in patients with low EGFR expression. Our work also highlights the relevance of epithelial to amoeboid transition in human tumours and the need to better target this process in the clinic

The level of caveolin-1 expression determines response to TGF-ß as a tumor 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-β) 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-β 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-β in hepatocytes. Whether this mechanism is relevant in hepatocarcinogenesis has not been explored yet. Here we analysed the TGF-β 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-β-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-β-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-β-induced EGFR transactivation and activation of the PI3K/AKT pathway. On the contrary, Huh7 cells, which do not respond to TGF-β 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-β from cytostatic to tumourigenic, which could have clinical meaning in patient stratification

Clathrin switches transforming growth factor-β 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-beta (TGF-beta) 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-beta-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-beta and EGFR signalling in vitro. Bioinformatic analysis was used to determine the effect of CLTC and TGEB1 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-beta 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-beta-induced apoptosis and upregulation of its pro-apoptotic target NOX4. Autocrine TGF-beta signalling in invasive HCC cells upregulates CLTC expression, switching its role to pro-tumorigenic. A positive correlation between TGEB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGEB1 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-beta pathways. CLTC expression in human HCC samples could help select patients that would benefit from TGF-beta-targeted therapy. Lay summary: Clathrin heavy-chain expression increases during liver tumorigenesis in humans (CLTC) and mice (Mc), altering the cellular response to TGF-beta in favour of anti-apoptotic/pro-tumorigenic signals. A positive correlation between TGEB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGEB1 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-beta. (C) 2019 European Association for the Study of the Liver. Published by Elsevier B

Famílies botàniques de plantes medicinals

Facultat de Farmàcia, Universitat de Barcelona. Ensenyament: Grau de Farmàcia, Assignatura: Botànica Farmacèutica, Curs: 2013-2014, Coordinadors: Joan Simon, Cèsar Blanché i Maria Bosch.Els materials que aquí es presenten són els recull de 175 treballs d’una família botànica d’interès medicinal realitzats de manera individual. Els treballs han estat realitzat per la totalitat dels estudiants dels grups M-2 i M-3 de l’assignatura Botànica Farmacèutica durant els mesos d’abril i maig del curs 2013-14. Tots els treballs s’han dut a terme a través de la plataforma de GoogleDocs i han estat tutoritzats pel professor de l’assignatura i revisats i finalment co-avaluats entre els propis estudiants. L’objectiu principal de l’activitat ha estat fomentar l’aprenentatge autònom i col·laboratiu en Botànica farmacèutica

New insights into the crosstalk between the TGF-β and the EGF Receptor pathways during liver regeneration and hepatocarcinogenesis

[eng] Transforming growth factor-beta (TGF-β) is an important growth suppressor in hepatocytes, inhibiting proliferation and inducing cell death. However, it also plays a role in other processes such as epithelial-to-mesenchymal transition (EMT), which contributes to tumour invasion and progression. Epidermal Growth Factor Receptor (EGFR) belongs to the kinase receptor family, and it is involved in cell proliferation, survival, differentiation and adhesion. Aiming to study the connection between both pathways, different animal and cellular models have been generated in our group. The in vivo model is based on transgenic mice that express specifically in hepatocytes a truncated form (in the kinase domain) of the human EGFR, acting as a dominant negative, decreasing the signalling of the pathway. On the other hand, it has been generated an in vitro model, decreasing the expression of EGFR by shRNA in different hepatocellular carcinoma (HCC) lines. This thesis focused, firstly, in the study of the crosstalk between both pathways during liver regeneration and HCC in the in vivo model. On the other hand, in the in vitro model, we analyzed this crosstalk related to the protumorigenic effects, such as adhesion and migration of tumour cells. Finally, we have evaluated the translational relevance of the results obtained, analyzing the gene expression in tissues from HCC patients. The results obtained in the in vivo model show that mice expressing the truncated form of EGFR in the hepatocytes present a delay in the process of liver regeneration after partial hepatectomy (PH) and in the appearance of tumours induced after injecting the carcinogen Diethylnitrosamine (DEN). Lack of EGFR catalytic activity promotes the overactivation of the TGF-β pathway during liver regeneration, correlating with the induction of the expression of cell cycle inhibitors, such as Cdkn1a (p21) and Cdkn2b (p15). EGFR pathway signalling is also required for the accumulation of lipid droplets and for the expression of a specific adipogenic profile during liver regeneration, suggesting a role for EGFR in the lipid metabolism in the liver. However, despite all these alterations, life of transgenic mice is not significantly compromised, and animals are able to fully regenerate the liver. Overactivation of the hepatocyte growth factor (HGF)/Met pathway and hyperplasia of the remaining cells could contribute to the success in the regenerative process. On the other hand, during DEN-induced tumorigenesis, delayed onset of tumours when EGFR catalytic activity is attenuated does not correlate with an increased Tgfb1 expression or overactivation of the Smads pathway. However, significant higher expression of Nox4, target of TGF-β and an inhibitor of cell proliferation in the liver, is observed. In the in vitro model, it is observed that the effects of TGF-β on the EMT phenotype and on the migratory capacities of HCC cells are heterogeneous. Thus, Hep3B cell line responds to it inducing a full EMT, whereas PLC/PRF/5 cell line undergoes partial EMT, increasing mesenchymal markers, but maintaining E-cadherin expression and cell-cell adhesion. EGFR silencing decreases cell-cell adhesions in PLC/PRF/5 and Hep3B. However, it is only able to decrease adhesion to matrix in the case of PLC/PRF/5, but not in Hep3B. All these results point that EGFR silencing induces a TGF-β-induced ameboid migration in PLC/ PRF/5 (but not in Hep3B), that correlates with higher actomyosin contractility and advantages in cell migration. Finally, analysis in tumour tissues of patients with HCC reveals that most of them present an overactivation of the TGF-β pathway and lower levels of EGFR. However, there is no correlation between the expression of TGFB1 and EGFR, suggesting that they could be regulated by independent mechanisms. Finally, the EMT-related genetic profile in HCC patients expressing high levels of TGFB1 is associated with EGFR expression, so that in tumours with low levels of EGFR, low expression of CDH1 and high expression of VIM is observed. Taken together, our results indicate that the EGFR pathway plays essential roles in the proliferation of hepatocytes under physiological conditions such as liver regeneration, regulating, among other processes, the cellular response to the suppressive effects of TGF-β. During carcinogenesis, at early stages it may contribute to the appearance of preneoplastic nodules, but once the tumour has formed, EGFR pathway may counteract some of the pro-migratory and invasive TGF-β signals. Therefore, it is necessary to deep into the mechanisms of crosstalk between both pathways, in order to target them therapeutically. The stratification of patients according to their gene expression may help in the application of the most appropriate therapies. This thesis provides information about some biomarkers that could be used for this purpose.[spa] El factor de crecimiento transformante-beta (TGF-β) es un importante supresor del crecimiento en hepatocitos, aunque desempeña otras funciones como la transición epitelio-mesénquima (EMT), contribuyendo a la invasión y progresión tumoral. El receptor del factor de crecimiento epidérmico (EGFR) está implicado en la proliferación, supervivencia celular, diferenciación y adhesión. Con el objetivo de estudiar la conexión entre ambas vías, en nuestro grupo se han generado diferentes modelos animales y celulares. El modelo in vivo basado en ratones transgénicos que expresan específicamente en los hepatocitos una forma truncada en el dominio quinasa del EGFR humano, actuando como dominante negativo, disminuyendo la señalización de la vía. Por otro lado, se han generado modelos in vitro, disminuyendo la expresión del EGFR mediante shRNA en diferentes líneas de carcinoma hepatocelular (CHC). Los resultados en el modelo in vivo muestran que los ratones transgénicos presentan un retraso en la regeneración hepática tras una hepatectomía parcial y en la aparición de tumores inducidos tras inyectar el carcinógeno dietilnitrosamina (DEN). En el model de regeneración hepática se observa una sobreactivación de la vía del TGF-β, un papel relevante para el EGFR en el metabolismo lipídico del hígado, y que estos animales son capaces de regenerar por completo el hígado gracias a mecanismos compensatorios tales como la sobreactivación de la vía del factor de crecimiento hepático (HGF)/Met y la hiperplasia de las células restantes. Por otro lado, en el retraso en la tumorogénesis inducida por DEN no correlaciona con una mayor expresión de Tgfb1, aunque sí se observa una significativa mayor expresión de Nox4, diana de TGF-β y un inhibidor de la proliferación celular en el hígado. En relación al modelo desarrollado in vitro, se observa que los efectos del TGF-β sobre el fenotipo de EMT y en las capacidades migratorias de las células de CHC son heterogéneos, induciendo en algunos casos una EMT total (en la línea celular Hep3B), y en otros una EMT parcial (en PLC/PRF/5). El silenciamiento del EGFR disminuye las adhesiones célula-célula en la línea celular PLC/PRF/5 y en Hep3B, pero solo disminuye la adhesión a la matriz en el caso de PLC/PRF/5. Todos estos efectos conllevan a una migración de tipo ameboide inducida por TGF-β en PLC/PRF/5, pero no en Hep3B. Por último, los análisis en tejidos tumorales de pacientes con HCC revelan que la mayoría de ellos presenta una activación de la vía del TGF-β y menores niveles de expresión del EGFR. No obstante, no hay correlación entre ellos, sugiriendo que podrian regularse por mecanismos independientes. En conjunto , nuestros resultados indican que la vía del EGFR juega papeles esenciales en la proiferación de los hepatocitos bajo situaciones fisiológicas como la regeneración hepática y la carcinogénesis. Durante la carcinogénesis, en etapas tempranas puede contribuir a la aparición de nódulos preneoplásicos, pero una vez se ha formado el tumor, la vía del EGFR podría contrarrestar algunas de las señales pro-migratorias e invasivas del TGF-β. Por consiguiente, es necesario profundizar en los mecanismos de conexión entre ambas vías, con el fin de atacarlas terapéuticamente

New insights into the crosstalk between the TGF-β and the EGF Receptor pathways during liver regeneration and hepatocarcinogenesis

Transforming growth factor-beta (TGF-β) is an important growth suppressor in hepatocytes, inhibiting proliferation and inducing cell death. However, it also plays a role in other processes such as epithelial-to-mesenchymal transition (EMT), which contributes to tumour invasion and progression. Epidermal Growth Factor Receptor (EGFR) belongs to the kinase receptor family, and it is involved in cell proliferation, survival, differentiation and adhesion. Aiming to study the connection between both pathways, different animal and cellular models have been generated in our group. The in vivo model is based on transgenic mice that express specifically in hepatocytes a truncated form (in the kinase domain) of the human EGFR, acting as a dominant negative, decreasing the signalling of the pathway. On the other hand, it has been generated an in vitro model, decreasing the expression of EGFR by shRNA in different hepatocellular carcinoma (HCC) lines. This thesis focused, firstly, in the study of the crosstalk between both pathways during liver regeneration and HCC in the in vivo model. On the other hand, in the in vitro model, we analyzed this crosstalk related to the protumorigenic effects, such as adhesion and migration of tumour cells. Finally, we have evaluated the translational relevance of the results obtained, analyzing the gene expression in tissues from HCC patients. The results obtained in the in vivo model show that mice expressing the truncated form of EGFR in the hepatocytes present a delay in the process of liver regeneration after partial hepatectomy (PH) and in the appearance of tumours induced after injecting the carcinogen Diethylnitrosamine (DEN). Lack of EGFR catalytic activity promotes the overactivation of the TGF-β pathway during liver regeneration, correlating with the induction of the expression of cell cycle inhibitors, such as Cdkn1a (p21) and Cdkn2b (p15). EGFR pathway signalling is also required for the accumulation of lipid droplets and for the expression of a specific adipogenic profile during liver regeneration, suggesting a role for EGFR in the lipid metabolism in the liver. However, despite all these alterations, life of transgenic mice is not significantly compromised, and animals are able to fully regenerate the liver. Overactivation of the hepatocyte growth factor (HGF)/Met pathway and hyperplasia of the remaining cells could contribute to the success in the regenerative process. On the other hand, during DEN-induced tumorigenesis, delayed onset of tumours when EGFR catalytic activity is attenuated does not correlate with an increased Tgfb1 expression or overactivation of the Smads pathway. However, significant higher expression of Nox4, target of TGF-β and an inhibitor of cell proliferation in the liver, is observed. In the in vitro model, it is observed that the effects of TGF-β on the EMT phenotype and on the migratory capacities of HCC cells are heterogeneous. Thus, Hep3B cell line responds to it inducing a full EMT, whereas PLC/PRF/5 cell line undergoes partial EMT, increasing mesenchymal markers, but maintaining E-cadherin expression and cell-cell adhesion. EGFR silencing decreases cell-cell adhesions in PLC/PRF/5 and Hep3B. However, it is only able to decrease adhesion to matrix in the case of PLC/PRF/5, but not in Hep3B. All these results point that EGFR silencing induces a TGF-β-induced ameboid migration in PLC/ PRF/5 (but not in Hep3B), that correlates with higher actomyosin contractility and advantages in cell migration. Finally, analysis in tumour tissues of patients with HCC reveals that most of them present an overactivation of the TGF-β pathway and lower levels of EGFR. However, there is no correlation between the expression of TGFB1 and EGFR, suggesting that they could be regulated by independent mechanisms. Finally, the EMT-related genetic profile in HCC patients expressing high levels of TGFB1 is associated with EGFR expression, so that in tumours with low levels of EGFR, low expression of CDH1 and high expression of VIM is observed. Taken together, our results indicate that the EGFR pathway plays essential roles in the proliferation of hepatocytes under physiological conditions such as liver regeneration, regulating, among other processes, the cellular response to the suppressive effects of TGF-β. During carcinogenesis, at early stages it may contribute to the appearance of preneoplastic nodules, but once the tumour has formed, EGFR pathway may counteract some of the pro-migratory and invasive TGF-β signals. Therefore, it is necessary to deep into the mechanisms of crosstalk between both pathways, in order to target them therapeutically. The stratification of patients according to their gene expression may help in the application of the most appropriate therapies. This thesis provides information about some biomarkers that could be used for this purpose.El factor de crecimiento transformante-beta (TGF-β) es un importante supresor del crecimiento en hepatocitos, aunque desempeña otras funciones como la transición epitelio-mesénquima (EMT), contribuyendo a la invasión y progresión tumoral. El receptor del factor de crecimiento epidérmico (EGFR) está implicado en la proliferación, supervivencia celular, diferenciación y adhesión. Con el objetivo de estudiar la conexión entre ambas vías, en nuestro grupo se han generado diferentes modelos animales y celulares. El modelo in vivo basado en ratones transgénicos que expresan específicamente en los hepatocitos una forma truncada en el dominio quinasa del EGFR humano, actuando como dominante negativo, disminuyendo la señalización de la vía. Por otro lado, se han generado modelos in vitro, disminuyendo la expresión del EGFR mediante shRNA en diferentes líneas de carcinoma hepatocelular (CHC). Los resultados en el modelo in vivo muestran que los ratones transgénicos presentan un retraso en la regeneración hepática tras una hepatectomía parcial y en la aparición de tumores inducidos tras inyectar el carcinógeno dietilnitrosamina (DEN). En el model de regeneración hepática se observa una sobreactivación de la vía del TGF-β, un papel relevante para el EGFR en el metabolismo lipídico del hígado, y que estos animales son capaces de regenerar por completo el hígado gracias a mecanismos compensatorios tales como la sobreactivación de la vía del factor de crecimiento hepático (HGF)/Met y la hiperplasia de las células restantes. Por otro lado, en el retraso en la tumorogénesis inducida por DEN no correlaciona con una mayor expresión de Tgfb1, aunque sí se observa una significativa mayor expresión de Nox4, diana de TGF-β y un inhibidor de la proliferación celular en el hígado. En relación al modelo desarrollado in vitro, se observa que los efectos del TGF-β sobre el fenotipo de EMT y en las capacidades migratorias de las células de CHC son heterogéneos, induciendo en algunos casos una EMT total (en la línea celular Hep3B), y en otros una EMT parcial (en PLC/PRF/5). El silenciamiento del EGFR disminuye las adhesiones célula-célula en la línea celular PLC/PRF/5 y en Hep3B, pero solo disminuye la adhesión a la matriz en el caso de PLC/PRF/5. Todos estos efectos conllevan a una migración de tipo ameboide inducida por TGF-β en PLC/PRF/5, pero no en Hep3B. Por último, los análisis en tejidos tumorales de pacientes con HCC revelan que la mayoría de ellos presenta una activación de la vía del TGF-β y menores niveles de expresión del EGFR. No obstante, no hay correlación entre ellos, sugiriendo que podrian regularse por mecanismos independientes. En conjunto , nuestros resultados indican que la vía del EGFR juega papeles esenciales en la proiferación de los hepatocitos bajo situaciones fisiológicas como la regeneración hepática y la carcinogénesis. Durante la carcinogénesis, en etapas tempranas puede contribuir a la aparición de nódulos preneoplásicos, pero una vez se ha formado el tumor, la vía del EGFR podría contrarrestar algunas de las señales pro-migratorias e invasivas del TGF-β. Por consiguiente, es necesario profundizar en los mecanismos de conexión entre ambas vías, con el fin de atacarlas terapéuticamente

The level of caveolin-1 expression determines response to TGF-ß as a tumor 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-β) 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-β 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-β in hepatocytes. Whether this mechanism is relevant in hepatocarcinogenesis has not been explored yet. Here we analysed the TGF-β 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-β-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-β-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-β-induced EGFR transactivation and activation of the PI3K/AKT pathway. On the contrary, Huh7 cells, which do not respond to TGF-β 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-β from cytostatic to tumourigenic, which could have clinical meaning in patient stratification

NADPH oxidase 4 (Nox4) deletion accelerates liver regeneration in mice

Liver is a unique organ in displaying a reparative and regenerative response after acute/chronic damage or partial hepatectomy, when all the cell types must proliferate to re-establish the liver mass. The NADPH oxidase NOX4 mediates Transforming Growth Factor-beta (TGF-β) actions, including apoptosis in hepatocytes and activation of stellate cells to myofibroblasts. Aim of this work was to analyze the impact of NOX4 in liver regeneration by using two mouse models where Nox4 was deleted: 1) general deletion of Nox4 (NOX4-/-) and 2) hepatocyte-specific deletion of Nox4 (NOX4hepKO). Liver regeneration was analyzed after 2/3 partial hepatectomy (PH). Results indicated an earlier recovery of the liver-to-body weight ratio in both NOX4-/- and NOX4hepKO mice and an increased survival, when compared to corresponding WT mice. The regenerative hepatocellular fat accumulation and the parenchyma organization recovered faster in NOX4 deleted livers. Hepatocyte proliferation, analyzed by Ki67 and phospho-Histone3 immunohistochemistry, was accelerated and increased in NOX4 deleted mice, coincident with an earlier and increased Myc expression. Primary hepatocytes isolated from NOX4 deleted mice showed higher proliferative capacity and increased expression of Myc and different cyclins in response to serum. Transcriptomic analysis through RNA-seq revealed significant changes after PH in NOX4-/- mice and support a relevant role for Myc in a node of regulation of proliferation-related genes. Interestingly, RNA-seq also revealed changes in the expression of genes related to activation of the TGF-β pathway. In fact, levels of active TGF-β1, phosphorylation of Smads and levels of its target p21 were lower at 24 h in NOX4 deleted mice. Nox4 did not appear to be essential for the termination of liver regeneration in vivo, neither for the in vitro hepatocyte response to TGF-β1 in terms of growth inhibition, which suggest its potential as therapeutic target to improve liver regeneration, without adverse effects.This work was supported by grants from: 1) Agencia Estatal de Investigación and Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (MICINN), Spain, cofounded by FEDER funds/European Regional Development Fund – a way to build Europe- (SAF2015-64149-R and RTI2018-094079-B-I00 to I.F.; PT17/0009/0019 to A.E.-C.; Programa Operativo FEDER: Plurirregional de España (POPE) 2014–2020 and Catalunya 2014–2020 to CNAG-CRG authors; 2) NIH 2R01DK083283 (to N.J.T.). M.H.-I. was recipient of a predoctoral grant from IDIBELL-Oncobell Program. D.C-D. was recipient of a pre-doctoral grant from the FPI program. J.L-L. was recipient of Boehringer Ingelheim Fonds Travel grant. The CIBEREHD, National Biomedical Research Institute on Liver and Gastrointestinal Diseases, is funded by the Instituto de Salud Carlos III, Spain. We thank the Generalitat de Catalunya through the CERCA Programme and the Departament de Salut and Departament d’Empresa i Coneixement, the Centro de Excelencia Severo Ochoa and the MICINN to the EMBL partnership for institutional suppor