DiFranco Kristina M, Mulligan Jennifer K, Sumal Aman S, Diamond Gill
Department of Oral Biology, University of Florida, Gainesville, FL 32610, United States, United States.
Department of Otolaryngology, Head and Neck Surgery, Medical University of South Carolina, Charleston, SC 29425, United States.
J Steroid Biochem Mol Biol. 2017 Oct;173:323-332. doi: 10.1016/j.jsbmb.2017.01.013. Epub 2017 Jan 24.
Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which often leads to protein misfolding and no CFTR surface localization. This then leads to chronic airway infections, inflammation, and tissue damage. Although vitamin D has been explored as a therapy to treat CF due to its antimicrobial-inducing and anti-inflammatory properties, the effect of 1,25-dihydroxyvitamin D (1α,25(OH)D) on CFTR directly has not been studied. We treated cultured healthy and diseased bronchial epithelial cells (BEC) with 10nM 1α,25(OH)D for 6 and 24h and found that 1α,25(OH)D increases both mRNA and protein CFTR levels using RT-qPCR, flow cytometry and fluorescence immunohistochemistry. Treatment of CF cells with 10nM 1α,25(OH)D led to an increase in both total and surface CFTR expression, suggesting 1α,25(OH)D could be used to increase properly localized CFTR in airway cells. To determine if BEC could convert the more clinically relevant cholecalciferol to 25OHD, cultured non-CF and CF BECs were treated with a range of cholecalciferol concentrations, and 25OHD levels were quantified by ELISA. We found that 25OHD levels increased in a concentration-dependent manner. Treatment of BEC with 10μM cholecalciferol led to increases in both CYP24A1 and CFTR mRNA levels, even when added to the apical surface of cells grown in an air-liquid interface, suggesting that topical administration of vitamin D could be used therapeutically. To demonstrate this in vivo, we intranasally delivered 1μM 1α,25(OH)D into mice. After 6h, we observed induction of both Cyp24A1 and CFTR expression in the tracheas of treated mice. The major findings of this study are that vitamin D can be converted to the active form when topically administered to the airway, and this could be used to increase CFTR levels in patients with CF. This could potentially be useful as an adjunctive therapy, together with newly developed CF treatments.
囊性纤维化（CF）是一种常染色体隐性疾病，由囊性纤维化跨膜传导调节因子（CFTR）基因突变引起，该突变常导致蛋白质错误折叠且CFTR无法定位到细胞表面。这进而导致慢性气道感染、炎症和组织损伤。尽管维生素D因其抗菌和抗炎特性已被探索用于治疗CF，但1,25 - 二羟基维生素D（1α,25(OH)D）对CFTR的直接作用尚未得到研究。我们用10nM的1α,25(OH)D处理培养的健康和患病支气管上皮细胞（BEC）6小时和24小时，通过逆转录定量聚合酶链反应（RT - qPCR）、流式细胞术和荧光免疫组织化学发现，1α,25(OH)D可增加CFTR的mRNA和蛋白质水平。用10nM的1α,25(OH)D处理CF细胞导致总CFTR和表面CFTR表达均增加，这表明1α,25(OH)D可用于增加气道细胞中定位正确的CFTR。为了确定BEC是否能将临床相关性更高的胆钙化醇转化为25OHD，用一系列胆钙化醇浓度处理培养的非CF和CF BEC，并通过酶联免疫吸附测定（ELISA）对25OHD水平进行定量。我们发现25OHD水平呈浓度依赖性增加。用10μM胆钙化醇处理BEC导致CYP24A1和CFTR mRNA水平均增加，即使将其添加到气液界面生长的细胞顶端表面时也是如此，这表明局部应用维生素D可用于治疗。为了在体内证明这一点，我们将1μM的1α,25(OH)D经鼻给药至小鼠体内。6小时后，我们观察到处理过的小鼠气管中Cyp24A1和CFTR表达均被诱导。本研究的主要发现是，维生素D局部应用于气道时可转化为活性形式，这可用于增加CF患者的CFTR水平。这可能作为一种辅助治疗方法，与新开发的CF治疗方法一起发挥作用。
