FOXJ1 Protein, Decreased leads to Motile Cilia Number/Length, Decreased

Foxj1 overexpression failed to promote ciliogenesis in mouse polarized epithelial cell lines and primary cultured alveolar epithelial cells (You et al., 2004). Also, the overexpression of Foxj1 in wild-type airway epithelial cells did not enhance the total number of ciliated cells. However, delivery of Foxj1 to null cells resulted in cilia formation (You et al., 2004).

Complete removal of FOXJ1 by means of homologous recombination in mouse embryonic stem cells resulted in absence of cilia in mouse airways (as well as in other typically multiciliated tissues such as oviduct, haploid sperm, choroid plexus and epithelial cells of the brain but not in embryonic node) (Brody et al., 2000; Chen J. et al., 1998).

Newly fertilized zebrafish eggs were injected with antisense morpholino oligonucleotides designed to block Foxj1a protein translation. Motile cilia numbers were severely reduced in Kupffer’s vesicle (KV), the floor plate and pronephric ducts 14 and 24 hpf (Yu et al., 2008).

Downregulation of Xenopus FoxJ1 produced a dose-dependent defect in skin cilia formation. When 20 ng or 40 ng morpholino oligonucleotides were injected, cilia formed, but were reduced in number and shortened in length. After injection of 75 ng morpholino oligos, most cilia were lost. Cilia length decreased from ~11 microns to 4 microns (Stubbs et al., 2008). Morpholino oligo knockdown of zebrafish FoxJ1 caused a two-fold decrease in the number of KV cilia and a 3.5-fold decrease in the average length of KV cilia (Stubbs et al., 2008).

RNAi against Schmidtea mediterranea foxJ1-4 substantially reduced the expression levels of foxJ1-4 which lead to almost complete loss of motile cilia (Vij et al., 2012).

In the presence of CSE, in FOXJ1 overexpressing human airway epithelial cells the average cilia length was significantly higher (5.2 μm) than in lentivirus-control–infected cells (4.1 μm) (Brekman et al., 2014). CSE was obtained from one Marlboro Red commercial cigarette bubbled in 12.5 ml of differentiation medium that was then 0.2 mm pore filtered. The absorbance was measured at 320 nm on a spectrophotometer and the optical density of 1 was defined as 100%. Homozygous FOXJ1 mutant mice were obtained by mating foxj1^+/- male and female animals. The explanted trachea of the foxj1^-/- mice harbors no motile cilia in contrast to wild-type trachea (Gomperts et al., 2004).

Exposure of differentiated human bronchial epithelial cells to 10% CSE decreased FOXJ1 expression by about 40% at 24 h and 70% at 72 h exposure. 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−1; 35 mL per each puff of 2 s duration with a volume of 0.5 cm above the filter. The CS solution was 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. Exposure to CSE concentration- and time-dependently reduced the average number of cells with cilia motility, which was significant after 3 days of incubation with CSE at 2.5% (about 30% inhibition), and reached a maximum of about 75% inhibition versus control after 7 days of incubation with CSE at 10% (Milara et al., 2012). Roflumilast N-oxide at 2 nM or 1 µM concentration-dependently prevented the decrease in the expression levels of Foxj1 mRNA and protein following 3 days of exposure of differentiated bronchial epithelial cells to CSE at 10%. Concurrently, roflumilast N-oxide partly prevented the loss in cells with cilia motility (Milara et al., 2012).

Electroporation using negative control (GFP-only) plasmid resulted in 45±1.4% (mean±s.e.m.) GFP+ ciliated cells in mouse trachea organ culture. FOXJ1 significantly increased the percentage of GFP+ ciliated cells to 68±3.6% (1.51-fold) (Johnson et al., 2018). 

14- or 24-hr treatment with antisense morpholino oligonucleotides designed to block Foxj1a protein translation results in absence of motile cilia in zebrafish (Yu et al., 2008).

Xenopus embryos were injected with FOXJ1 morpholino oligos at two-cell stage (1.5 h of embryo life) and the embryos were analyzed at stage 26 (1 day, 5 h and 30 min of embryo life) for cilia phenotype (Stubbs et al., 2008).

Schmidtea mediterranea worms received three feedings of foxJ1-4 RNAi (2 days in between feeds) and were analyzed for cilia phenotype 14 days after the last feed. RNAi against Schmidtea mediterranea foxJ1-4 substantially reduced the expression levels of foxJ1-4 which lead to almost complete loss of motile cilia (Vij et al., 2012).

Exposure of differentiated human bronchial epithelial cells to CSE concentration- and time-dependently reduced the average number of cells with cilia motility, which was significant after 3 days of incubation with CSE at 2.5% (about 30% inhibition), and reached a maximum of about 75% inhibition versus control after 7 days of incubation with CSE at 10% (Milara et al., 2012).

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