Liu Yan-Nien, Kang Bean-Bu, Chen J H
Graduate Institute of Human Genetics, Tzu Chi University, Hualien, Taiwan.
Oncogene. 2004 Jan 8;23(1):278-88. doi: 10.1038/sj.onc.1207022.
Osteopontin (OPN) is a secreted glycoprotein produced by osteoclasts, macrophages, T cells, hematopoietic cells, and vascular smooth muscle cells. It contributes to macrophage homing and cellular immunity. It also mediates neovascularization, inhibits apoptosis, and plays important roles in extracellular matrix remodeling and angiogenesis. These properties are also characteristics of metastatic cancer cells. Consequently, the OPN gene was found to be upregulated among various metastatic cancer cells. This suggests that OPN is involved in tumor metastasis. How the OPN gene is upregulated in metastatic cancer cells remains to be illustrated. Thus, we investigated the transcriptional activation of the OPN promoter in the human metastatic cancer cell line A2058. We cloned the OPN promoter, and serial deletion analysis of the OPN promoter showed that the region between -170 and -127 may act as an enhancer to control the OPN gene in metastatic tumor cells. This region was found to contain overlapped AML-1 and C/EBP binding site motifs. Gel-mobility-shift assays using the A2058 nuclear extract and AML-1a or C/EBPalpha (CCAAT/enhancer binding protein alpha) recombinant protein indicated that these two transcription factors can bind to the overlapped AML-1 /C/EBP binding site motifs on the OPN regulatory sequence from -147 to -127. Surprisingly, the gel-shift experiments did not show supershift complex formation between AML-1 and C/EBPalpha. Functional analysis showed that the C/EBPalpha was more potent than the complex of AML-1 and its cofactor CBFbeta to upregulate the OPN promoter. In addition, AML-1 and C/EBPalpha did not exhibit transactivation additively or synergistically. Our results suggest that AML-1 and C/EBPalpha play an important role in the upregulation of the OPN gene in metastatic tumor cells.
骨桥蛋白（OPN）是一种由破骨细胞、巨噬细胞、T细胞、造血细胞和血管平滑肌细胞分泌的糖蛋白。它有助于巨噬细胞归巢和细胞免疫。它还介导新血管形成，抑制细胞凋亡，并在细胞外基质重塑和血管生成中发挥重要作用。这些特性也是转移性癌细胞的特征。因此，发现OPN基因在各种转移性癌细胞中上调。这表明OPN参与肿瘤转移。OPN基因在转移性癌细胞中如何上调仍有待阐明。因此，我们研究了人转移性癌细胞系A2058中OPN启动子的转录激活情况。我们克隆了OPN启动子，对OPN启动子的系列缺失分析表明，-170至-127之间的区域可能作为增强子来控制转移性肿瘤细胞中的OPN基因。发现该区域包含重叠的AML-1和C/EBP结合位点基序。使用A2058核提取物以及AML-1a或C/EBPα（CCAAT/增强子结合蛋白α）重组蛋白进行的凝胶迁移率变动分析表明，这两种转录因子可以结合到OPN调控序列上从-147至-127重叠的AML-1/C/EBP结合位点基序。令人惊讶的是，凝胶迁移实验未显示AML-1和C/EBPα之间形成超迁移复合物。功能分析表明，C/EBPα比AML-1及其辅因子CBFβ的复合物更有效地上调OPN启动子。此外，AML-1和C/EBPα未表现出相加或协同的反式激活作用。我们的结果表明AML-1和C/EBPα在转移性肿瘤细胞中OPN基因的上调中起重要作用。
