Elloumi Houda Zghal, Holland Steven M
Immunopathogenesis Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, CRC B3-4141, MSC 1684, Bethesda, MD 20892, USA.
Mol Immunol. 2008 Jan;45(1):204-17. doi: 10.1016/j.molimm.2007.04.023. Epub 2007 Aug 20.
Cationic antimicrobial peptides play important roles in host defense, linking innate and adaptive immunity. hCAP18, the only human antimicrobial cathelicidin, consists of a conserved N-terminal cathelin-like domain and a C-terminal peptide, LL-37. Expression is regulated during myeloid differentiation, and tightly controlled during infection and inflammation, suggesting active regulation. Using 5' RACE (rapid amplification of cDNA ends), multiple transcription initiation sites were identified, as well as new splice variants leading to novel augmentations of hCAP18 amino acid composition in bone marrow but not peripheral blood neutrophils. Having expressed hCAP18 promoter constructs in cell lines, we found that full-length (-1739) and truncated (-978) promoter constructs had lower luciferase activities than 5'UTR deletion constructs. Transient transfection of progressively deleted constructs in the non-permissive K562 cell line led us to identify a negative regulatory element within the 53 bp immediately upstream of the ATG of hCAP18. Additionally, transient transfection of 5' deletion constructs identified a positive regulatory element within the 101 bases 5' of promoter sequence containing two GT-boxes. Negative and positive regulatory elements within the hCAP18 gene promoter provide new insights into the possible molecular basis of myeloid gene expression.
阳离子抗菌肽在宿主防御中发挥重要作用，连接天然免疫和适应性免疫。hCAP18是唯一的人类抗菌cathelicidin，由一个保守的N端类cathelin结构域和一个C端肽LL-37组成。其表达在髓系分化过程中受到调控，在感染和炎症期间受到严格控制，提示存在主动调控。利用5' RACE（cDNA末端快速扩增）技术，鉴定出多个转录起始位点以及新的剪接变体，这些变体导致hCAP18在骨髓而非外周血中性粒细胞中的氨基酸组成出现新的增加。在细胞系中表达hCAP18启动子构建体后，我们发现全长（-1739）和截短（-978）的启动子构建体的荧光素酶活性低于5'UTR缺失构建体。在非允许性K562细胞系中对逐步缺失的构建体进行瞬时转染，使我们在hCAP18的ATG上游紧邻的53 bp内鉴定出一个负调控元件。此外，对5'缺失构建体进行瞬时转染，在启动子序列5'端包含两个GT-box的101个碱基内鉴定出一个正调控元件。hCAP18基因启动子内的正负调控元件为髓系基因表达可能的分子基础提供了新的见解。
