Myokai F, Takashiba S, Lebo R, Amar S
Boston University, Department of Periodontology and Oral Biology, School of Dental Medicine, Boston, MA 02118, USA.
Proc Natl Acad Sci U S A. 1999 Apr 13;96(8):4518-23. doi: 10.1073/pnas.96.8.4518.
Lipopolysaccharide (LPS) is a potent stimulator of monocytes and macrophages, causing secretion of tumor necrosis factor alpha (TNF-alpha) and other inflammatory mediators. Given the deleterious effects to the host of TNF-alpha, it has been postulated that TNF-alpha gene expression must be tightly regulated. The nature of the nuclear factor(s) that control TNF-alpha gene transcription in humans remains obscure, although NF-kappaB has been suggested. Our previous studies pertaining to macrophage response to LPS identified a novel DNA-binding domain located from -550 to -487 in the human TNF-alpha promoter that contains transcriptional activity, but lacks any known NF-kappaB-binding sites. We have used this DNA fragment to isolate and purify a 60-kDa protein binding to this fragment and obtained its amino-terminal sequence, which was used to design degenerate probes to screen a cDNA library from THP-1 cells. A novel cDNA clone (1.8 kb) was isolated and fully sequenced. Characterization of this cDNA clone revealed that its induction was dependent on LPS activation of THP-1 cells; hence, the name LPS-induced TNF-alpha factor (LITAF). Inhibition of LITAF mRNA expression in THP-1 cells resulted in a reduction of TNF-alpha transcripts. In addition, high level of expression of LITAF mRNA was observed predominantly in the placenta, peripheral blood leukocytes, lymph nodes, and the spleen. Finally, chromosomal localization using fluorescence in situ hybridization revealed that LITAF mapped to chromosome 16p12-16p13.3. Together, these findings suggest that LITAF plays an important role in the activation of the human TNF-alpha gene and proposes a new mechanism to control TNF-alpha gene expression.
脂多糖（LPS）是单核细胞和巨噬细胞的强效刺激剂，可导致肿瘤坏死因子α（TNF-α）及其他炎症介质的分泌。鉴于TNF-α对宿主具有有害作用，据推测TNF-α基因表达必须受到严格调控。尽管有人提出核因子κB（NF-κB），但控制人类TNF-α基因转录的核因子的性质仍不清楚。我们之前关于巨噬细胞对LPS反应的研究在人类TNF-α启动子中鉴定出一个新的DNA结合结构域，位于-550至-487，该结构域具有转录活性，但缺乏任何已知的NF-κB结合位点。我们利用这个DNA片段分离并纯化了一种与该片段结合的60 kDa蛋白，并获得了其氨基末端序列，该序列被用于设计简并探针以筛选THP-1细胞的cDNA文库。分离出一个新的cDNA克隆（1.8 kb）并进行了全序列测定。对该cDNA克隆的表征显示，其诱导依赖于LPS对THP-1细胞的激活；因此，将其命名为LPS诱导的TNF-α因子（LITAF）。抑制THP-1细胞中LITAF mRNA的表达会导致TNF-α转录本减少。此外，主要在胎盘、外周血白细胞、淋巴结和脾脏中观察到LITAF mRNA的高水平表达。最后，使用荧光原位杂交进行染色体定位显示，LITAF定位于16号染色体p12 - 16p13.3。总之，这些发现表明LITAF在人类TNF-α基因的激活中起重要作用，并提出了一种控制TNF-α基因表达的新机制。
