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Oxidative Stress leads to FOXJ1 Protein, Decreased
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding||Point of Contact||Author Status||OECD Status|
|Oxidative Stress Leading to Decreased Lung Function via Decreased FOXJ1||adjacent||Karsta Luettich (send email)||Open for comment. Do not cite|
|Homo sapiens||Homo sapiens||High||NCBI|
Life Stage Applicability
|All life stages|
Key Event Relationship Description
Oxidative stress (such as that caused by cigarette smoke exposure or irradiation) leads to decreased forkhead box J1 (FOXJ1) gene and protein expression, as well as to decreased FOXJ1 target gene expression (Brekman et al., 2014; Garcia-Arcos et al., 2016; Ishikawa and Ito, 2017; Milara et al., 2012; Valencia-Gattas et al., 2016). FOXJ1 is a key factor of multiple motile cilia assembly in the respiratory airways (Zhou and Roy, 2015). Thus oxidative stress blocks the multiple ciliogenesis program in the airway epithelium.
Evidence Collection Strategy
Evidence Supporting this KER
Cigarette smoke-induced oxidative stress downregulates FOXJ1 expression at both the gene and protein levels in human lung cells in vitro (Milara et al., 2012; Brekman et al., 2014; Valencia-Gattas et al., 2016; Ishikawa and Ito, 2017). Oxidative stress induced by human respiratory syncytial virus reduces FOXJ1 mRNA levels, which can be restored by treatment with antioxidants or the phosphodiesterase 4 inhibitor roflumilast N-oxide (Akaike et al., 1990; Geiler et al., 2010; Mata et al., 2012). In mice, thoracic irradiation results in free radical generation and subsequent reduction in FOXJ1 mRNA expression (Bernard et al., 2012). Many genes that are transcriptionally regulated by FOXJ1 are also downregulated following exposure to cigarette smoke, which implies a reduction in FOXJ1 transcriptional activity (Brekman et al., 2014).
The negative association between cigarette smoke exposure and FOXJ1 levels in airways was shown in multiple studies and can be estimated as a strong linkage. Yet, the notion that oxidative stress as a result of cigarette smoke exposure is leading to decreased FOXJ1 levels is not well demonstrated. As a complex mixture of thousands of chemicals, cigarette smoke exposure could lead to reduced FOXJ1 levels via different routes. Indirect evidence, such as antioxidant molecules that restore cigarette smoke exposure-reduced FOXJ1 levels, as well evidences from other oxidative stress generating insults that decrease FOXJ1 levels add confidence to this KER. However, studies showing a link between oxidative stress generating agents and reduced FOXJ1 levels are scarce. Collectively, the empirical evidence and uncertainties of the linkage imply a moderate ranking for the KER.
Whole cigarette smoke exposed normal human bronchial epithelial cells (HBECs) had significantly lower FoxJ1 protein levels than air-exposed controls (by immunofluorescence). In addition, FOXJ1 mRNA levels were reduced in whole smoke-exposed differentiating and differentiated HBECs (by TaqMan quantitative RT-PCR)(Valencia-Gattas et al., 2016).
Treatment with cigarette smoke extract (CSE) significantly down-regulated FOXJ1 mRNA and protein levels in differentiating human airway basal cells in air–liquid interface (ALI) cultures (by TaqMan qRT-PCR and Western blot analysis) (Brekman et al., 2014).
Treatment with cigarette smoke extract (CSE) reduced FOXJ1 gene and protein expression in differentiated HBECs through an IL13-mediated mechanism (by RT-qPCR and Western blot analysis) (Milara et al., 2012). Treatment with roflumilast N-oxide (which reduced intracellular reactive oxygen species levels) prevented FOXJ1 loss in CSE-treated cells (Milara et al., 2012).
Exposure of 3D co-cultures of HBECs and fibroblasts to whole cigarette smoke decreased FOXJ1 gene expression in a concentration-dependent manner (by TaqMan quantitative RT-PCR) (Ishikawa and Ito, 2017).
Human respiratory syncytial virus (RSV) infections involve reactive oxygen intermediates (ROIs) that cause cellular damage (Akaike et al., 1990; Mata et al., 2012). Treatment with the free radical scavenger N-acetylcysteine (NAC) reduced the RSV inflammatory response (Geiler et al., 2010). RSV infection reduced FOXJ1 gene expression (by RT-PCR), which was restored in a dose-dependent manner by NAC treatment (Mata et al., 2012). In another study, FOXJ1 mRNA levels were consistently low after RSV infection and were restored with roflumilast N-oxide (Mata et al., 2013).
Thoracic irradiation reduced FOXJ1 mRNA levels in mouse lungs (Bernard et al., 2012). Irradiation causes excessive levels of free radicals and associated lipid peroxidation, damage to DNA, proteins, leading to wide-spread cellular damage (Azzam et al., 2012; Koc et al., 2003; Rodrigues-Moreira et al., 2017; Shirazi et al., 2013).
The expression of cilia-related transcription factor genes, including FOXJ1, RFX2, and RFX3 was significantly down-regulated by CSE treatment. The expression of cilia motility and structural integrity genes, including DNAI1, DNAH5, DNAH9, DNAH10, DNAH11, and SPAG6 was also significantly down-regulated by CSE treatments (Brekman et al., 2014). Many of these genes (RFX2, RFX3, DNAI1, DNAH9, DNAH11, SPAG6) are transcriptionally regulated by FOXJ1 (Causal biological network database, 2019). The downregulation of FOXJ1-controlled genes infer reduced FOXJ1 transcription factor activity. Indeed, overexpression of FOXJ1 led to partial restoration of CSE treatment-induced downregulation of cilia-related genes (Brekman et al., 2014).
Uncertainties and Inconsistencies
Schamberger et al. did not find any alterations in FOXJ1 mRNA levels or FOXJ1 target gene (DNAI1, DNALI1, SPAG6, TEKT1) transcription upon exposure of HBECs to 2.5% or 5% CSE for 28 days. However, in this study, cigarette smoke exposure reduced ciliated cell numbers (Schamberger et al., 2015).
The evidences listed suggest several mechanisms on how oxidative stress could lead to decreased FOXJ1 levels, including EGFR-, MCIDAS- or IL-13-mediated mechanisms. Most of the studies, however, do not corroborate on how oxidative stress mechanistically leads to reduced FOXJ1 levels. Since there are several other factors (GMNC, NOTCH, ULK4 etc.) known to regulate FOXJ1 levels, further pathways might be involved in passing the oxidative stress signal to FOXJ1.
Known modulating factors
Quantitative Understanding of the Linkage
High oxidative stress causes reduction in FOXJ1 levels measured at 24 h and for up to 15 days after exposure to an oxidative stress-causing agent. The data on cigarette smoke-reduced FOXJ1 levels are convincing. Indirect evidence such as antioxidants restoring CS-reduced FOXJ1 levels suggest that oxidative stress plays a major role in the CS-induced effects (Milara et al., 2012). However, given the complexity of the CS mixtures (Baumung et al., 2016), we cannot exclude that factors other than oxidative stress are involved in FOXJ1 level reduction. Other sources of oxidative stress such as RSV infection or GY radiation reduce FOXJ1 levels to similar degree as CS-caused FOXJ1 reduction. Based on the available evidence, we classify the quantitative understanding of this KER as moderate.
Normal HBECs were exposed to whole cigarette smoke from 3R4F research grade cigarettes using the Vitrocell® VC 10® Smoking Robot (35-mL puff volume, 2 s duration and 1 min between puffs or air as a control). Differentiated cells were exposed every 2 d for 5 d (3 exposures), and samples were collected 48 h after treatment. Differentiating cells were exposed 3 times per week to smoke from 1 cigarette, and samples were collected after 14, 21 and 27 days. FOXJ1 protein and mRNA levels decreased 2.5-fold in differentiated and 2-fold in differentiating normal HBECs. There was a significantly lower percentage of FOXJ1-positive cells following whole smoke exposure at day 27 (4.3 +/- 4.2% vs. 13.0 +/- 7.3%, air) (Valencia-Gattas et al., 2016).
CSE was obtained from one Marlboro Red commercial cigarette bubbled in 12.5 mL of differentiation medium that was then filtered (0.2-µm pore filter). The absorbance was measured at 320 nm on a spectrophotometer, and the optical density of 1 was defined as 100% CSE. HBECs were differentiated at the air-liquid interface while being exposed to 0, 3, and 6% CSE between days 5 and 28. Treatment with 3% and 6% CSE reduced FOXJ1 mRNA levels to approx. 65% and 55% of control levels, respectively. Treatment of differentiating cultures with 3% CSE reduced FOXJ1 protein levels by 2-fold by day 28 (Brekman et al., 2014).
The smoke of one 2R4F research cigarette was bubbled into a flask containing 25 mL of pre-warmed (37°C) differentiation medium using a respiratory pump model (Harvard Apparatus Rodent Respirator 680, Harvard Apparatus, Holliston, MA, USA) that generates three puffs/min; 35 mL per each puff of 2-s duration with a volume of 0.5 cm above the filter. The solution was then filtered (0.22 µm pore size) to remove particles and the tar phase. The resulting sterile solution was defined as 100% CSE and used within 30 min of preparation. Treatment of differentiated human bronchial epithelial cells with 10% CSE decreased FOXJ1 expression by about 40% at 24 h and 70% at 72 h exposure (Milara et al., 2012).
3R4F reference cigarettes were smoked in accordance with the ISO smoking protocol (35-mL puffs of 2 s each minute). Whole cigarette smoke, generated by a VC10 smoking robot, was released into a mixing device in 2.8-s exhaust and diluted with humidified clean air at 1.0 L/min dilution flow. Diluted smoke was introduced into the CULTEX RFS module and guided into the exposure chamber (5 mL/min) using a vacuum pump to expose 3D co-cultures of HBECs and fibroblasts. FOXJ1 mRNA levels were reduced to 60% and 40% of the control after exposure to CS from 1 or 4 cigarettes, respectively (Ishikawa and Ito, 2017).
RSV infection elicits reactive oxygen intermediate-mediated effects manifested by changes in the expression of NRF2 and HMOX-1 genes (approx. 6- and 9-fold increase at day 15 post-RSV infection, respectively), H2O2 generation (7-fold increase in intracellular levels at day 15 post-infection) and severe reduction in total antioxidant capacity. These data together indicate the presence of oxidative stress following infection which leads to decreased FOXJ1 mRNA levels (ca. 25% of control at 15 days post-RSV infection (Mata et al., 2012) and ca. 45% of control at 10 days after RSV infection (Mata et al., 2013).
FOXJ1 mRNA levels were reduced by 50% in murine lungs 14 days after thoracic irradiation at 15 Gy (Bernard et al., 2012).
Whole cigarette smoke exposure from one 3R4F research grade cigarette using the Vitrocell® VC 10® Smoking Robot (35-mL puff volume, 2-s duration and 1 min between puffs or air as a control) once a day on alternate days for 5 days decreased FOXJ1 mRNA levels by 2.5-fold in differentiated HBECs (Valencia-Gattas et al., 2016).
Whole cigarette smoke exposure from one 3R4F research grade cigarette using the Vitrocell® VC 10® Smoking Robot (35-mL puff volume, 2-s duration and 1 min between puffs or air as a control) for 3 times per week for 4 weeks (27 days) decreased FOXJ1 mRNA levels by 2-fold in differentiating HBECs (Valencia-Gattas et al., 2016).
Treatment of differentiating HBECs (between days 5 and 28 of the air-liquid interface) with 3% CSE reduced FOXJ1 protein levels by 2-fold by day 28. Treatment of differentiating HBECs with 6% CSE reduced FOXJ1 protein levels by approx. 55% by day 28 (Brekman et al., 2014).
Treatment of differentiated human bronchial epithelial cells with 10% CSE decreased FOXJ1 expression by about 40% at 24 h and ca. 70% at 72 h (Milara et al., 2012).
Repeated exposure of 3D bronchial epithelial cultures to whole smoke of 4 cigarettes (every other day, treatment started on ALI culture day 7) resulted in a 2.5-fold decrease of FOXJ1 mRNA levels by day 21 (Ishikawa and Ito, 2017).
At 15 days post-RSV infection, FOXJ1 mRNA levels were 4-fold reduced compared to untreated samples (Mata et al., 2012). In another study from the same group, FOXJ1 mRNA levels were reduced to 45% of the FOXJ1 levels in uninfected sample at 10 days post-RSV infection (Mata et al., 2013).
At 7 and 14 days after 15 GY thoracic irradiation, FOXJ1 mRNA levels were reduced to approx. 70% and 50% of controls, respectively (Bernard et al., 2012).
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
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Azzam, E.I., Jay-Gerin, J.P., and Pain, D. (2012). Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury. Cancer Lett. 327, 48-60.
Baumung, C., Rehm, J., Franke, H., and Lachenmeier, D.W. (2016). Comparative risk assessment of tobacco smoke constituents using the margin of exposure approach: the neglected contribution of nicotine. Sci. Rep. 6, 35577.
Bernard, M.E., Kim, H., Rajagopalan, M.S., Stone, B., Salimi, U., Rwigema, J.C., et al. (2012). Repopulation of the irradiation damaged lung with bone marrow-derived cells. In Vivo 26, 9-18.
Brekman, A., Walters, M.S., Tilley, A.E., and Crystal, R.G. (2014). FOXJ1 prevents cilia growth inhibition by cigarette smoke in human airway epithelium in vitro. Am. J. Respir. Cell Mol. Biol. 51, 688-700.
Garcia-Arcos, I., Geraghty, P., Baumlin, N., Campos, M., Dabo, A.J., Jundi, B., et al. (2016). Chronic electronic cigarette exposure in mice induces features of COPD in a nicotine-dependent manner. Thorax 71, 1119-1129.
Ishikawa, S., and Ito, S. (2017). Repeated whole cigarette smoke exposure alters cell differentiation and augments secretion of inflammatory mediators in air-liquid interface three-dimensional co-culture model of human bronchial tissue. Toxicol. In Vitro 38, 170-178.
Koc, M., Taysi, S., Buyukokuroglu, M.E., and Bakan, N. (2003). Melatonin protects rat liver against irradiation-induced oxidative injury. J. Radiat. Res. 44, 211-215.
Mata, M., Martinez, I., Melero, J.A., Tenor, H., and Cortijo, J. (2013). Roflumilast inhibits respiratory syncytial virus infection in human differentiated bronchial epithelial cells. PLoS One 8, e69670.
Mata, M., Sarrion, I., Armengot, M., Carda, C., Martinez, I., Melero, J.A., et al. (2012). Respiratory syncytial virus inhibits ciliagenesis in differentiated normal human bronchial epithelial cells: effectiveness of N-acetylcysteine. PloS One 7, e48037.
Milara, J., Armengot, M., Bañuls, P., Tenor, H., Beume, R., Artigues, E., et al. (2012). Roflumilast N-oxide, a PDE4 inhibitor, improves cilia motility and ciliated human bronchial epithelial cells compromised by cigarette smoke in vitro. Br. J. Pharmacol. 166, 2243-2262.
Rodrigues-Moreira, S., Moreno, S.G., Ghinatti, G., Lewandowski, D., Hoffschir, F., Ferri, F., et al. (2017). Low-Dose Irradiation Promotes Persistent Oxidative Stress and Decreases Self-Renewal in Hematopoietic Stem Cells. Cell Rep. 20, 3199-3211.
Schamberger, A.C., Staab-Weijnitz, C.A., Mise-Racek, N., and Eickelberg, O. (2015). Cigarette smoke alters primary human bronchial epithelial cell differentiation at the air-liquid interface. Sci. Rep. 5, 8163.
Shirazi, A., Mihandoost, E., Ghobadi, G., Mohseni, M., and Ghazi-Khansari, M. (2013). Evaluation of radio-protective effect of melatonin on whole body irradiation induced liver tissue damage. Cell J. 14, 292-297.
Valencia-Gattas, M., Conner, G.E., and Fregien, N.L. (2016). Gefitinib, an EGFR Tyrosine Kinase inhibitor, Prevents Smoke-Mediated Ciliated Airway Epithelial Cell Loss and Promotes Their Recovery. PloS One 11, e0160216.
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