RESUMO
BACKGROUND AND AIMS: Netrin-1 displays protumoral properties, though the pathological contexts and processes involved in its induction remain understudied. The liver is a major model of inflammation-associated cancer development, leading to HCC. APPROACH AND RESULTS: A panel of cell biology and biochemistry approaches (reverse transcription quantitative polymerase chain reaction, reporter assays, run-on, polysome fractionation, cross linking immunoprecipitation, filter binding assay, subcellular fractionation, western blotting, immunoprecipitation, stable isotope labeling by amino acids in cell culture) on in vitro-grown primary hepatocytes, human liver cell lines, mouse samples and clinical samples was used. We identify netrin-1 as a hepatic inflammation-inducible factor and decipher its mode of activation through an exhaustive eliminative approach. We show that netrin-1 up-regulation relies on a hitherto unknown mode of induction, namely its exclusive translational activation. This process includes the transfer of NTN1 (netrin-1) mRNA to the endoplasmic reticulum and the direct interaction between the Staufen-1 protein and this transcript as well as netrin-1 mobilization from its cell-bound form. Finally, we explore the impact of a phase 2 clinical trial-tested humanized anti-netrin-1 antibody (NP137) in two distinct, toll-like receptor (TLR) 2/TLR3/TLR6-dependent, hepatic inflammatory mouse settings. We observe a clear anti-inflammatory activity indicating the proinflammatory impact of netrin-1 on several chemokines and Ly6C+ macrophages. CONCLUSIONS: These results identify netrin-1 as an inflammation-inducible factor in the liver through an atypical mechanism as well as its contribution to hepatic inflammation.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Camundongos , Humanos , Animais , Receptor 2 Toll-Like , Fatores de Crescimento Neural/metabolismo , Receptor 3 Toll-Like , Receptor 6 Toll-Like , Proteínas Supressoras de Tumor/metabolismo , Inflamação/metabolismo , Anti-Inflamatórios , RNA Mensageiro , Aminoácidos , Receptores de NetrinaRESUMO
It is becoming increasingly evident that reactive oxygen species (ROS) have critical roles as "second messengers" in cell signaling. In B cells, ROS can be generated either as a byproduct of mitochondrial respiration, as a result of the endoplasmic reticulum stress response induced by high production of Igs, or by the activation of NADPH oxidase (NOX) complexes. Having previously shown that costimulation of B cells via TLR 9 and the TLR-related receptor RP105 drives maturation of human peripheral blood B cells into Ig-producing cells, we aimed to study the role of ROS generated during this vital process. To this end, the ROS levels were either reduced by the NOX inhibitor VAS2870 or by the ROS scavenger N-acetyl cysteine (NAC). We revealed that TLR9/RP105-mediated stimulation of human B cells involved a rapid activation of NOX. Moreover, VAS2870 blocked the TLR9/RP105-induced B cell activation and thereby all Ig production. Importantly, we showed that ROS targeted by NAC was selectively required for IgG but not for IgM production. The endoplasmic reticulum stress response in the TLR9/RP105-stimulated cells was higher in IgG+ than in IgG- cells and was reduced by NAC in IgG+ cells only. Of note, we revealed that substantially higher levels of IgG than IgM were produced per cell and that IgG+ cells produced significantly higher ROS levels than IgG- cells. Taken together, our results imply that NAC-targeted ROS may be particularly important for sustaining the high Ig production in IgG+ B cells.