Angiogenic endothelium-specific nestin expression is enhanced by the first intron of the nestin gene
Angiogenic endothelium-specific nestin expression is enhanced by the first intron of the nestin gene
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DOI：
10.1038/labinvest.3700186
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发表时间：
2004-12-01
影响因子：
5
通讯作者：
Takeuchi, T
中科院分区：
文献类型：
--
作者：
Aihara, M;Sugawara, K;Takeuchi, T
巢蛋白是神经干细胞和胶质母细胞瘤中大量表达的中间纤维。nestin基因有4个外显子和3个内含子，神经细胞特异性表达受第二个内含子调控。我们先前曾报道，巢蛋白总是检测到肿瘤内皮细胞在胶质瘤，即使肿瘤细胞是阴性的巢蛋白。在本研究中，我们进一步证实了巢蛋白免疫染色的肿瘤内皮细胞的各种常见的癌症，包括肺癌，胃癌，结肠癌和宫颈癌。我们使用人脐静脉内皮细胞（HUVECs）和人胶质母细胞瘤衍生的U251细胞研究了内皮特异性调节剂。在荧光素酶报告基因测定中，第一个内含子加5'上游启动子（5' UP）产生最高活性，其次是5' UP，第二个内含子加5' UP。然而，HUVEC提取物的测定值远低于U251细胞提取物。尽管在第一内含子、第二内含子或普遍活性的CAG启动子下，绿色荧光蛋白表达在所有U251细胞上均为阳性，但即使在第一内含子下，HUVEC中的荧光也仅限于少数细胞。这种差异来自HUVECs的生长特性，其通过接触抑制而表现出生长停滞。我们发现巢蛋白的表达是特异性的增殖内皮细胞，通过使用在血管母细胞瘤的增殖标记和原位杂交。使用内皮管形成试验，酪氨酸激酶结构域缺失的VEGF受体KDR有效地废除了第一内含子下的管形成。我们认为第一内含子增强了肿瘤内皮细胞巢蛋白的表达。
Nestin is a member of intermediate filaments abundantly expressed in neural stem cells and glioblastomas. The nestin gene has four exons and three introns, and neural cell-specific expression is regulated by the second intron. We previously reported that nestin was invariably detected in the tumor endothelium in gliomas even though tumor cells were negative for nestin. In the present study, we further confirmed nestin immunostaining in tumor endothelium of a variety of common cancers, including lung, stomach, colon, and cervical carcinomas. We examined an endothelium-specific regulator using human umbilical vein endothelial cells (HUVECs) and human glioblastoma-derived U251 cells. In a luciferase reporter assay, the first intron plus 5' upstream promoter (5' UP) gave the highest activity, followed by 5' UP, and the second intron plus 5' UP. However, the assay values were much lower by HUVEC extracts than by U251 cell extracts. Although green fluorescent protein expression was positive over all U251 cells under either the first intron, second intron, or ubiquitously active CAG promoter, the fluorescence in HUVECs was limited to a few cells even under the first intron. This difference came from the growth feature of HUVECs which exhibit growth arrest by contact inhibition. We found that the nestin expression was specific to proliferative endothelium, by using proliferation markers in hemangioblastomas and in situ hybridization. Using an endothelial tube formation assay, tyrosine kinase domain-deleted VEGF receptor KDR effectively abolished the tube formation under the first intron. We suggest that the nestin expression in tumor endothelium is enhanced by the first intron.