The Sp1 transcription factor contributes to the tumor necrosis factor-induced expression of the angiogenic factor thymidine phosphorylase in human colon carcinoma cells
The Sp1 transcription factor contributes to the tumor necrosis factor-induced expression of the angiogenic factor thymidine phosphorylase in human colon carcinoma cells
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DOI：
10.1038/sj.onc.1206030
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发表时间：
2002-12-05
期刊：
影响因子：
8
通讯作者：
Schwartz, EL
中科院分区：
文献类型：
--
作者：
Zhu, GH;Lenzi, M;Schwartz, EL
胸苷磷酸化酶（TP，又称血小板源性内皮细胞生长因子，PD-ECGF）是一种血管生成因子，对内皮细胞具有趋化作用，在体内可诱导新生血管形成。TP在人类实体肿瘤中经常过表达，其表达与肿瘤微血管密度增加、侵袭和转移以及患者生存期缩短有关。本研究发现，肿瘤坏死因子（tumor necrosis factor, TNFalpha）可诱导WiDr结肠癌细胞系的TP活性提高100倍。TNFalpha是活化巨噬细胞的分泌产物，具有间接的血管生成活性。TP活性的增加伴随着TP mRNA水平的增加，而mRNA的稳定性没有增加。米霉素（一种富含gc序列的dna结合转录抑制剂）可降低tnfalpha诱导的TP mRNA水平。在荧光素酶报告基因构建中，将上游TP序列瞬时转染WiDr，证实TNFa对转录的调控作用。缺失分析指出TP启动子的两个区域具有tnfalpha诱导和基础表达的调控元件，它们分别包含3个和1个sp1转录因子家族的一致结合位点。另外一个区域只参与TP的基础表达，包含3个Sp1位点。当四个Sp1位点中的三个发生点突变时，tnfalpha诱导的TP表达减少，这些位点被认为有助于基础表达和tnfalpha诱导的表达。电泳迁移率转移实验进一步证实了Sp1核与这三个位点的结合。用TNFalpha处理的细胞也增加了sp1结合活性。这些研究确立了Sp1在结肠癌WiDr细胞中调控血管生成因子TP表达的作用。
Thymidine phosphorylase (TP; also known as platelet-derived endothelial cell growth factor, PD-ECGF) is an angiogenic factor that is chemotactic for endothelial cells and has been found to induce neovascularization in vivo. TP is frequently overexpressed in human solid tumors, where its expression has been correlated with increased tumor microvessel density, invasion, and metastasis, and shorter patient survival. In this report, TP activity in the WiDr colon carcinoma cell line was found to be induced 100-fold by tumor necrosis factor (TNFalpha), a secretory product of activated macrophages that has indirect angiogenic activities. Increased TP activity was accompanied by increased TP mRNA levels and without an increase in mRNA stability. TNFalpha-induced TP mRNA levels were reduced by mithramycin, a DNA-binding transcription inhibitor specific for GC-rich sequences. Transcriptional regulation by TNFa was confirmed by transient transfection of WiDr with upstream TP sequences in a luciferase reporter construct. Deletion analysis of the reporter pinpointed two regions of the TP promoter with regulatory elements for both TNFalpha-inducible and basal expression, and they contained, respectively, three and one consensus binding sites for the Sp1-family of transcription factors. One additional region contributed only to basal TP expression, and it contained three Sp1 sites. TNFalpha-induced TP expression decreased when point mutations were made in three of the four Sp1 sites postulated to contribute to both basal and TNFalpha-inducible expression. Electrophoretic mobility shift assays further demonstrated binding of nuclear Sp1 to these three sites. Sp1-binding activity was also increased in cells treated with TNFalpha. These studies establish a role for Sp1 in the regulation of expression of the angiogenic factor TP in colon cancer WiDr cells.