Follistatin Protects Pulmonary Epithelial Cells Against Silica Nanoparticle-induced Oxidative Stress

Silica nanoparticle (SiO2 NP) are increasingly being used for diverse industrial and biomedical applications. The wide use of SiO2 NP has raised serious concerns about their safety for human health. The respiratory system is considered to be one of the main routes by which SiO2 NP access human body. Therefore, the respiratory toxicity of SiO2 NP draws great concern. SiO2 NP induces the generation of reactive oxygen species (ROS) in airway epithelial cells and macrophages. Elevated ROS triggers cellular oxidative stress responses which are mainly mediated by the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. Nrf2 is a transcription factor that induces the expression of protective proteins including antioxidant enzymes, detoxification enzymes, and stress-response proteins. However, the regulation of other SiO2 NP-responsive genes and their functions in SiO2 NP response remain largely unknown.Follistatin (FST) was identified as a secretory protein that binds and inactivates transforming growth factor (TGF)-β family members. FST participates in a variety of processes such as cell growth, development, differentiation and secretion. Our recent evidence showed that under glucose deprivation or hypoxia, the expression level of FST is up-regulated and the protein promotes cell survival. However, the function of FST in cellular response to SiO2 NP treatment remains elusive.To study the role of FST in SiO2 NP generated ROS, we firstly detected whether SiO2 NP treatment could induce FST expression in mice lung tissue. The mRNA level of FST dramatically increased at 1 and 2 day after SiO2 NP instillation. Consistently, immunoblotting and immunohistochemistry staining showed that the protein level of FST increased at 1 and 7 day after SiO2 NP treatment. SiO2 NP also dramatically up-regulated FST mRNA and protein level in A549 cells. These data indicated that FST is a SiO2 NP-responsive gene.The higher level of FST mRNA may result from either transcriptional activation or post-transcriptional regulation. We first examined the stability of FiST mRNA. Actinomycin D was used to block de novo RNA synthesis, and then the persistence of the existing FST mRNA was measured. Our results revealed that the stability of the FST mRNA did not change during SiO2 NP treatment. Therefore, we detected changes in FST gene promoter activity using a luciferase reporter. Data showed that the transcriptional activity of FST promoter region was significantly induced under SiO2 NP treatment. We further detected the levels of Ac-H3 (K9/18) and H3K4me2 which are active gene markers using chromatin immunoprecipitation (ChIP). Data showed that SiO2 NP significantly increased both Ac-H3(K9/18) and H3K4me2 levels at FST promoter region, further supporting that SiO2 NP up-regulates FST expression at the transcriptional level.The transcriptional factor Nrf2 controls stress gene expression during SiO2 NP treatment. To determine the role of endogenous Nrf2 in regulating FST expression, Nrf2 levels was decreased by siRNA. Knockdown of Nrf2 resulted in significant reduction of FST mRNA and protein expression under normal condition and SiO2 NP treatment. By analyzing the FST gene proximal promoter, we identified a putative antioxidant response element (ARE), which is a consensus Nrf2-binding sequence. Data revealed that luciferase activity of the wild-type FST promoter reporter was significantly reduced following knockdown of Nrf2, indicating that FST promoter activity is Nrf2-dependent. However, deletion of ARE region completely abolished Nrf2 effect. ChIP assays data unveiled enrichment of FST promoter region following immunoprecipitation with the Nrf2 antibody. SiO2 NP treatment significantly increased Nrf2 binding to FST promoter. These data indicated that FST gene is a direct target of Nrf2.Since FST expression was up-regulated in response to SiO2 NP, we suspected that FST has a protective role. We therefore determined cellular apoptosis by annexin V-FITC staining method. SiO2 NP treatment markedly increased cell apoptosis in control siRNA-transfected cells, and elimination of FST enhanced the apoptotic tendency, indicating FST has a protective role in apoptosis. Obvious TUNEL-positive staining was observed in SiO2 NP-treated mice lungs, indicating that SiO2 NP exposure induced cell apoptosis in mice lung tissue. The number of TUNEL-positive cells increased in FST knockdown cells. These data suggested that FST protects cells against SiO2 NP-induced cellular toxicity both in vitro and in vivo.The toxic effect of SiO2 NP is mainly mediated by particle-induced oxidative stress. We therefore determined whether FST exerts its protective function through inhibiting ROS production during SiO2 NP exposure. SiO2 NP treatment increased total ROS level in A549 cells. Knockdown of FST further increased ROS level while overexpression of FST decreased ROS level, indicating an inhibitory role of FST in SiO2 NP-induced ROS production. Furthermore, our data showed that the mRNA levels of NADPH oxidase 1 (NOX1) and NOX5 significantly increased during SiO2 NP treatment. Knockdown of FST increased while overexpression of FST decreased the expression of NOX1 and NOX5 under both control and SiO2 NP treatment. These data suggested that FST inhibits NOX1 and NOX5 expression to regulate cytosolic ROS production.In summary, the major findings of our study include.1) SiO2 NP induced endogenous expression of FST both in mouse lungs and in cultured lung epithelial cells; 2) SiO2 NP activated FST transcription through Nrf2; 3) FST inhibited SiO2 NP-induced apoptosis; 4) FST inhibited ROS production triggered by SiO2 NP. These findings demonstrated a protective role of FST and might provide a better understanding of the interaction between SiO2 NP and biological systems. These findings point to a protective strategy for respiratory system to overcome SiO2 NP-induced adverse effect.