gms | German Medical Science

Objectives: Previous researches have demonstrated 1, 25-dihydroxyvitamine D3 (VD3) is essential for the maintenance of alveolar epithelial barrier integrity. The lack of VD3 in human serum is more predisposed to result in acute lung injuries (ALI) and chronic obstructive pulmonary diseases (COPD) caused by pathogen infections. The positive effect of VD3 on the property of alveolar epithelial barrier has been verified in our prior study. However, the molecular mechanisms involved in this process are still not fully clarified. Our proteomic analysis on the VD3 (10 nM) treated alveolar type II (ATII)-like epithelial cell line, NCI-H441 cells, discovered the significant impact of VD3 on the production of doublecortin-like kinase 1 (DCLK1), which is a serine/threonine kinase and classified in the family of microtubule-associated proteins. DCLK1 has been revealed to participate in the maintenance of barrier integrity of epithelial cells and the regulation of inflammatory response during injuries. Therefore, DCLK1 could be a critical molecule induced by VD3 in alveolar epithelial cell to improve the barrier resistance against infection and injuries. The effect of VD3 on the expression of DCLK1 in alveolar epithelial cells was more deeply investigated in this study.

Methods: NCI-H441 cells were seeded apically on the semi-permeable Transwell® filters. The epithelial barrier properties were determined by trans-epithelial electrical resistance (TEER) measurement with EVOM equipment. The cells were cultured for 4 and 7 days. VD3 (10 nM) treatment was implemented on day 3. Alternatively, the bacterial endotoxin lipopolysaccharide (LPS) treatment (100 ng/mL or 10 ug/mL) was implemented from day 5 to day 7 for 48 hours. The protein composition was analyzed through Tandem Mass Spectrometry (MS/MS). The expression levels of DCLK1 in NCI-H441 cells were evaluated by real-time PCR, Western-Blot, and ELISA. Accordantly, its expression was also morphologically analyzed in NCI-H441 cells through immunofluorescent staining and microscopy.

Results and conclusion: VD3 (10 nM) significantly improve the alveolar epithelial barrier properties. The expression of DCLK1 was prominently stimulated by VD3 (10 nM) in both genetic and protein levels, which was consistent with its morphological fluorescent detection. The results revealed the remarkable effect of VD3 on the expression of DCLK1 in alveolar epithelial cells. The discovery is newly and firstly demonstrated based on the researches up to date in our knowledge. The role and function of DCLK1 associated with the maintenance of alveolar epithelial integrity will be further explored in our following investigations.