Oxidative Stress leads to FOXJ1 Protein, Decreased

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.  

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).

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