Zheng Jiamao, Ather Jennifer L, Sonstegard Tad S, Kerr David E
Lactation and Mammary Gland Biology Group, Department of Animal Science, 213 Terrill Hall, University of Vermont, Burlington, VT 05405, USA.
Gene. 2005 Jun 20;353(1):107-17. doi: 10.1016/j.gene.2005.04.016.
The concentration of lactoferrin in bovine milk is dramatically increased in response to infection. The high levels of lactoferrin may have a role in the prevention of microbial infection of the mammary gland. However, molecular mechanisms of how the lactoferrin gene is regulated in the mammary gland in response to infection remain unknown. In this study, we isolated and characterized the 5' flanking region of the bovine lactoferrin gene. An 8.2 kilobase (kb) fragment of the bovine lactoferrin gene, containing 4.4 kb of 5' flanking region, exon 1, intron 1, and exon 2, was isolated from a bovine genomic library on two overlapping bacterial artificial chromosome (BAC) clones. Sequence analysis of the isolated lactoferrin gene revealed that the promoter region contains a high GC content, a non-canonical TATA box, multiple stimulating protein 1 (SP1)/GC elements, and other putative binding sites for transcription factors including nuclear factor-kappaB (NF-kappaB), activator protein 1 (AP1), signal transducer and activator of transcriptions 3 and 5 (STAT3 and STAT5), and steroid hormone receptors. To demonstrate that the isolated promoter is functional, 4.4 kb of 5' flanking region was inserted upstream from the firefly luciferase gene and the chimeric construct was transiently transfected into murine mammary epithelial cells. Transfection studies showed that the basal promoter activity is quite potent, being similar in strength to that of the simian virus 40 (SV40) promoter/enhancer. In addition, a 24-h treatment with Escherichia coli lipopolysaccharide (LPS) significantly stimulated its activity up to 2.3-fold in a dose-dependent manner. Furthermore, promoter deletion analysis indicated that the sequence up to -543 was sufficient for basal activity, whereas the sequence up to -1029 was required for maximal basal activity. The basal activity of the promoter is affected by both positive regulatory regions (-2462/-1879 and -1029/-75) and a negative regulatory region (-1407/-1029). LPS-responsive regions of the promoter were localized to the region from -1029 to -543 containing one STAT3 site and two NF-kappaB sites, and the region from -4355 to -2462 containing three AP1 sites and six NF-kappaB sites. Taken together, our findings suggested that the lactoferrin promoter responds to infection via the NF-kappaB pathway.
牛乳中乳铁蛋白的浓度会因感染而显著增加。高水平的乳铁蛋白可能在预防乳腺的微生物感染中发挥作用。然而，乳铁蛋白基因在乳腺中如何响应感染进行调控的分子机制仍不清楚。在本研究中，我们分离并鉴定了牛乳铁蛋白基因的5'侧翼区域。从牛基因组文库的两个重叠细菌人工染色体（BAC）克隆中分离出一个8.2千碱基（kb）的牛乳铁蛋白基因片段，其包含4.4 kb的5'侧翼区域、外显子1、内含子1和外显子2。对分离出的乳铁蛋白基因进行序列分析表明，启动子区域富含GC，有一个非典型的TATA盒、多个刺激蛋白1（SP1）/GC元件以及其他转录因子的假定结合位点，包括核因子-κB（NF-κB）、激活蛋白1（AP1）、信号转导和转录激活因子3和5（STAT3和STAT5）以及类固醇激素受体。为了证明分离出的启动子具有功能，将4.4 kb的5'侧翼区域插入萤火虫荧光素酶基因上游，并将嵌合构建体瞬时转染到小鼠乳腺上皮细胞中。转染研究表明，基础启动子活性相当强，强度与猿猴病毒40（SV40）启动子/增强子相似。此外，用大肠杆菌脂多糖（LPS）处理24小时以剂量依赖的方式显著刺激其活性，最高可达2.3倍。此外，启动子缺失分析表明，-543之前的序列足以支持基础活性，而-1029之前的序列是最大基础活性所必需的。启动子的基础活性受到正调控区域（-2462/-1879和-1029/-75）和负调控区域（-1407/-1029）的影响。启动子的LPS响应区域定位于-1029至-543的区域，该区域包含一个STAT3位点和两个NF-κB位点，以及-4355至-2462的区域，该区域包含三个AP1位点和六个NF-κB位点。综上所述，我们的研究结果表明乳铁蛋白启动子通过NF-κB途径响应感染。
