Calcitriol modifies tight junctions, improves barrier function, and reduces TNF‐α‐induced barrier leak in the human lung‐derived epithelial cell culture model, 16HBE 14o‐
Elizabeth Rybakovsky, Katherine M Diguilio, Mary Carmen Valenzano, Sophie Geagan, Kaithlyn Pham, Ronald N Harty, James M Mullin
Physiological Reports, doi:10.14814/phy2.15592
Using the 16HBE 14o-human airway epithelial cell culture model, calcitriol (Vitamin D) was shown to improve barrier function by two independent metrics -increased transepithelial electrical resistance (TER) and reduced transepithelial diffusion of 14 C-D-mannitol (J m ). Both effects were concentration dependent and active out to 168 h post-treatment. Barrier improvement associated with changes in the abundance of specific tight junctional (TJ) proteins in detergent-soluble fractions, most notably decreased claudin-2. TNF-αinduced compromise of barrier function could be attenuated by calcitriol with a concentration dependence similar to that observed for improvement of control barrier function. TNFαinduced increases in claudin-2 were partially reversed by calcitriol. The ERK 1,2 inhibitor, U0126, itself improved 16HBE barrier function indicating MAPK pathway regulation of 16HBE barrier function. Calcitriol's action was additive to the effect of U0126 in reducing TNF-α -induced barrier compromise, suggesting that calcitriol may be acting through a non-ERK pathway in its blunting of TNF-α -induced barrier compromise. This was supported by calcitriol being without effect on pERK levels elevated by the action of TNF-α. Lack of effect of TNF-α on the death marker, caspase-3, and the inability of calcitriol to decrease the elevated LC3B II level caused by TNF-α, suggest that calcitriol's barrier improvement does not involve a cell death pathway. Calcitriol's improvement of control barrier function was not additive to barrier improvement induced by retinoic acid (Vitamin A). Calcitriol improvement and protection of airway barrier function could in part explain Vitamin D's reported clinical efficacy in COVID-19 and other airway diseases.
AUTHOR CONTRIBUTIONS
ACKNOWLEDGMENTS The authors are very grateful to Ms. Terri Olshefski and Ms. Elene Mironidis of the Editorial Office of the Lankenau Institute for Medical Research for their work in formatting and editing our manuscript for publication. The assistance of Ms. Elizabeth Newberry in proofreading is also gratefully acknowledged. We are very thankful to Dr. Mazen Hassan and Ms. Kari Heller for their help in obtaining needed reference material.
FUNDING INFORMATION Financial support for this research came in part from a research grant from the Sharpe-Strumia Research Foundation (JMM) and NIH grant AI139392 (RNH).
ETHICAL STATEMENT This Study used neither animal nor human subjects. All science was conducted in an ethical manner.
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