Abstract
BackgroundHyperoxia downregulates the tight junction (TJ) proteins of the alveolar epithelium and leads to barrier dysfunction. Previous study has showed that STE20/SPS1-related proline/alanine-rich kinase (SPAK) interferes with the intestinal barrier function in mice. The aim of the present study is to explore the association between SPAK and barrier function in the alveolar epithelium after hyperoxic exposure.MethodsHyperoxic acute lung injury (HALI) was induced by exposing mice to > 99% oxygen for 64 h. The mice were randomly allotted into four groups comprising two control groups and two hyperoxic groups with and without SPAK knockout. Mouse alveolar MLE-12 cells were cultured in control and hyperoxic conditions with or without SPAK knockdown. Transepithelial electric resistance and transwell monolayer permeability were measured for each group. In-cell western assay was used to screen the possible mechanism of p-SPAK being induced by hyperoxia.ResultsCompared with the control group, SPAK knockout mice had a lower protein level in the bronchoalveolar lavage fluid in HALI, which was correlated with a lower extent of TJ disruption according to transmission electron microscopy. Hyperoxia down-regulated claudin-18 in the alveolar epithelium, which was alleviated in SPAK knockout mice. In MLE-12 cells, hyperoxia up-regulated phosphorylated-SPAK by reactive oxygen species (ROS), which was inhibited by indomethacin. Compared with the control group, SPAK knockdown MLE-12 cells had higher transepithelial electrical resistance and lower transwell monolayer permeability after hyperoxic exposure. The expression of claudin-18 was suppressed by hyperoxia, and down-regulation of SPAK restored the expression of claudin-18. The process of SPAK suppressing the expression of claudin-18 and impairing the barrier function was mediated by p38 mitogen-activated protein kinase (MAPK).ConclusionsHyperoxia up-regulates the SPAK-p38 MAPK signal pathway by ROS, which disrupts the TJ of the alveolar epithelium by suppressing the expression of claudin-18. The down-regulation of SPAK attenuates this process and protects the alveolar epithelium against the barrier dysfunction induced by hyperoxia.
Highlights
Hyperoxia downregulates the tight junction (TJ) proteins of the alveolar epithelium and leads to bar‐ rier dysfunction
The resistance of the barrier (Rb) decreased significantly in the control group after hyperoxic exposure, but not in the p38 mitogenactivated protein kinases (MAPK) inhibitor group (Fig. 5f ). These findings indicate that SPS1-related proline/alanine-rich kinase (SPAK) modulates hyperoxia-induced barrier dysfunction and the suppression of claudin-18 via p38 MAPK in the alveolar epithelium
Downregulation of SPAK alleviates the phosphorylation of p38 and restores the expression of claudin-18, which protects the alveolar epithelium against the barrier dysfunction in hyperoxic acute lung injury (HALI) (Fig. 6)
Summary
Hyperoxia downregulates the tight junction (TJ) proteins of the alveolar epithelium and leads to bar‐ rier dysfunction. The aim of the present study is to explore the association between SPAK and barrier function in the alveolar epithelium after hyperoxic exposure. Hyperoxia results in the accumulation of inflammatory mediators within the lungs This process involves protein kinases such as serine-threonine kinase Akt, mitogenactivated protein kinases (MAPK), protein kinase C, and transcription factors such as NF-E2-related transcription factor 2 and nuclear factor-κB [5,6,7]. Transmembrane and peripheral proteins compose the tight junction (TJ) that attach cells tightly to their neighbors and form a barrier. Previous studies have shown that hyperoxia downregulates the expression of TJ proteins in the alveolar epithelium in HALI, which leads to barrier dysfunction [15,16,17]
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