SCCA1 在气道上皮细胞中与哮喘相关的生理事件中的作用，以及启动子变异对哮喘和基因功能的影响。

The role of SCCA1 in asthma related physiological events in the airway epithelium and the effect of promoter variants on asthma and gene function.

机构信息

Hacettepe University, Faculty of Science, Department of Biology, 06800 Ankara, Turkey.

出版信息

Respir Med. 2013 Mar;107(3):368-79. doi: 10.1016/j.rmed.2012.11.003. Epub 2012 Nov 28.

DOI:10.1016/j.rmed.2012.11.003

PMID:23199842

Abstract

BACKGROUND

Even though the systemic level of SCCA1, a serine protease inhibitor, was shown to be elevated in asthma, its physiological role is unknown.

OBJECTIVE

We sought to determine the effect of SCCA1 on apoptosis, cytokine expression and mucus production by A549 cells and define the effect of promoter variants on gene expression and association with asthma.

METHODS

SCCA levels were measured by ELISA. Promoter variants were determined by direct sequencing. 442 asthmatic children and 191 controls were genotyped by RFLP. The functional effect of the polymorphisms was assessed in transient transfection experiments using reporter constructs. A transcription factor ELISA was used for differential binding of GATA proteins to the variant region. The effects of SCCA1 on cytokine synthesis, mucus production and apoptosis were determined in A549 cells transfected with SCCA1 pcDNA vector. MUC5AC expression in A549 cells was determined with RT-PCR.

RESULTS

SCCA1 protein level was significantly higher in asthmatic children compared to healthy controls. Four polymorphisms SCCA1 promoter that were in linkage disequilibrium were associated with skin test positivity in asthmatic children and showed higher promoter activity and higher binding of GATA-2 and GATA-3 after IL-4 + IL-13 stimulation. IL-6, IL-8 levels were significantly higher in cells transfected with SCCA1 whereas RANTES increased only after IL-4 stimulation. Transfection of A549 cells with SCCA1 resulted in decreased MUC5AC expression and conferred protection against apoptosis.

CONCLUSION

Our results showed that SCCA1 has diverse effects on many of the cellular events that characterize asthma and its role extends beyond protease inhibition.

摘要

背景

尽管丝氨酸蛋白酶抑制剂 SCCA1 的系统水平在哮喘中升高，但它的生理作用尚不清楚。

目的

我们旨在确定 SCCA1 对 A549 细胞凋亡、细胞因子表达和黏液产生的影响，并确定启动子变体对基因表达的影响及其与哮喘的关联。

方法

通过 ELISA 测量 SCCA 水平。通过直接测序确定启动子变体。通过 RFLP 对 442 名哮喘儿童和 191 名对照进行基因分型。使用报告基因构建体在瞬时转染实验中评估多态性的功能效应。转录因子 ELISA 用于评估 GATA 蛋白对变体区域的差异结合。用 SCCA1 pcDNA 载体转染 A549 细胞，测定 SCCA1 对细胞因子合成、黏液产生和凋亡的影响。用 RT-PCR 测定 A549 细胞中 MUC5AC 的表达。

结果

与健康对照相比，哮喘儿童的 SCCA1 蛋白水平显著升高。与哮喘儿童皮肤试验阳性相关的 SCCA1 启动子的 4 种多态性呈连锁不平衡，并且在 IL-4+IL-13 刺激后表现出更高的启动子活性和更高的 GATA-2 和 GATA-3 结合。与转染细胞相比，转染 SCCA1 的细胞中 IL-6、IL-8 水平显著升高，而 RANTES 仅在 IL-4 刺激后增加。转染 A549 细胞的 SCCA1 导致 MUC5AC 表达降低，并对细胞凋亡具有保护作用。