Role of the NADPH Oxidase NOX4 in Liver Fibrosis and Hepatocarcinogenesis

Abstract

Reactive oxygen species (ROS) are short-lived, highly electrophilic molecules generated by the partial reduction of oxygen to form superoxide, hydrogen peroxide and hydroxyl radical as well as other secondary metabolites. For years, ROS have been considered as damaging molecules; however, in the last 20 years it has been proved that low levels of ROS can provoke transient and reversible modifications, leading to the regulation of signalling pathways.

Within the cell there are numerous potential sources of ROS. They can be produced as by-products of enzymatic processes, which is considered the case of mitochondrial ROS, but they can also be produced as primary species, which is the case of ROS from the NADPH oxidase (NOX) family of enzymes that has emerged in the last years as an important source of ROS in signal transduction. NOX-derived ROS have been implicated in regulation of cytoskeletal remodelling, gene expression, proliferation, differentiation, migration and cell death. The NOX family is composed of seven members, NOX1-5 and two dual oxidases (DUOX1-2), which share analogies in structure and catalytic function, since all of them are transmembrane flavoproteins that mediate the reduction of oxygen using NADPH as an electron donor. However, important differences in regulation and cellular functions are observed among them.

It has been reported that NOX1, NOX2 and NOX4 are expressed in the liver. Indeed, previous works from our group demonstrated that different NOX isoforms play different functions, sometimes even opposite roles, in hepatocytes.

In this work we analyse the role of the NADPH oxidase NOX4 in different physiological and pathological situations of the liver such as liver regeneration, liver fibrosis and hepatocarcinogenesis.

First, we describe the role of NOX4 during liver fibrosis. We show that Nox4 expression is up-regulated during liver fibrosis in two different mice models, and it is required for TGF-ß­induced transdifferentiation of hepatic stellate cells (HSC) to myofibroblasts, as well as for hepatocyte apoptosis.

Secondly, we analyse the relevance of the autocrine activation of the Transforming Growth Factor-beta (TGF-ß) pathway during hepatocarcinogenesis and its relation with NOX4. We show that high levels of autocrine TGF-ß provoke loss of epithelial characteristics, gain of mesenchymal features and resistance to TGF-ß suppressor effects, in different human hepatocellular carcinoma (HCC) cells. Surprisingly, there is an inverse correlation between autocrine TGF-ß and NOX4 expression in HCC cell lines.

Another important point in this work is the study of the role of NOX4 in the proliferative capacity of hepatocytes, as well as in their tumorigenic capacity. Results show that NOX4 is a negative regulator of proliferation in both untransformed hepatocytes and liver tumour cells. Moreover, its expression is down-regulated during liver regeneration after partial hepatectomy in mice.

Furthermore, Nox4 expression is also down-regulated during Diethylnitrosamine (DEN)­induced liver tumorigenesis in mice, and targeting knock-down of NOX4 in HCC cells confers them advantage for tumour progression in xenograft models in mice. Low levels of NOX4 are found in human HCC cell lines and in a relevant percentage of human liver tumour tissues.

Finally, we analyse the role of NOX4 in the metastatic capacity of HCC cell lines. We describe that down-regulation of NOX4 increases the invasive growth capacity of HCC cells when embedded in a pliable matrix, increasing both proliferation and invasion. Interestingly, low levels of NOX4 correlate with high actomyosin contractility, which provokes loss of epithelial parenchymal characteristics, decreasing both cell-to-cell contacts and cell-to-matrix adhesion, and increasing invasive capacity.

In summary, in the liver, NOX4 plays pro-fibrotic roles, mediating TGF-ß-induced HSC activation and hepatocyte death, but plays a tumour suppressor role during carcinogenesis, being a negative regulator of liver tumour growth and invasion.

La familia de las NADPH oxidasas (NOXes) se considera una de las principales fuentes de radicales libres de oxígeno (ROS) involucrados en señalización celular. Se ha descrito que en el hígado se expresan las isoformas NOX1, NOX2 y NOX4, y trabajos previos del grupo han demostrado que estas pueden jugar papeles diferentes e incluso antagónicos.

En este trabajo analizamos el papel de NOX4 en diferentes procesos fisiopatológicos del hígado.

Describimos la relevancia de NOX4 en la inducción de la fibrosis hepática, mediando los efectos del TGF-ß, tanto a nivel de transactivación de las células hepáticas estrelladas a miofibroblastos, como a nivel de inducción de apoptosis en hepatocitos.

También analizamos la relevancia de la vía del TGF-ß en los procesos de hepatocarcinogenesis. Niveles altos de TGF-ß autocrino provocan la pérdida de características epiteliales, la ganancia de características mesenquimales y la resistencia a los efectos del TGF-ß como supresor tumoral. Además mostramos una correlación inversa entre los niveles de expresión de TGF-ß y NOX4.

Por otro lado demostramos que NOX4 juega un papel como inhibidor del crecimiento tanto en células de hepatocarcinoma humano como en hepatocitos no transformados. Además, mostramos una caída en los niveles de NOX4 durante la regeneración hepática así como durante la hepatocarcinogenesis inducida por Dietilnitrosamina (DEN) en modelos murinos. También mostramos una mayor capacidad tumorigénica de las células cuando la expresión de NOX4 esta atenuada. Además, los niveles de NOX4 se encuentran disminuidos en muestras de tejidos humanos de hepatocarcinoma.

Finalmente, analizamos el papel de NOX4 en la capacidad metastásica de células hepatocarcinoma. Describimos que la disminución en los niveles de NOX4 incrementa la capacidad de crecimiento invasivo de las células en una matriz de colágeno. Además, niveles bajos de NOX4 correlacionan con una mayor contractilidad de la actomiosina y una destrucción de las estructuras parenquimáticas de las células, disminuyendo las adhesiones célula-célula y célula-matriz extracelular, incrementando la capacidad invasiva de las mismas.

En resumen, en este trabajo describimos que en el hígado, NOX4 tiene un papel pro­fibrótico mediando la activación de las HSC a miofibroblastos y la muerte de los hepatocitos inducidas por TGF-ß, pero también tiene un papel como supresor tumoral durante la carcinogénesis, por ser un regulador negativo del crecimiento tumoral y de la invasión.