Molecular characterization of NF-HEV, a nuclear factor preferentially expressed in human high endothelial venules
Molecular characterization of NF-HEV, a nuclear factor preferentially expressed in human high endothelial venules
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DOI：
10.1016/s0002-9440(10)63631-0
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发表时间：
2003-07-01
影响因子：
6
通讯作者：
Girard, JP
中科院分区：
文献类型：
--
作者：
Baekkevold, ES;Roussigné, M;Girard, JP
淋巴细胞归巢到次级淋巴组织和慢性炎症病变是由循环细胞和高内皮微静脉（HEV）的特化内皮之间的多步相互作用指导的。本研究采用抑制性消减杂交（SSH）技术，通过比较新鲜纯化的HEV内皮细胞（HEVECs）和鼻息肉来源的微血管内皮细胞（PMECs），筛选新的HEV基因。通过这种方法，我们克隆了第一个优先在HEVEC中表达的核因子，命名为HEV核因子（NF-HEV）。虚拟北方和Western印迹分析显示，与人脐静脉内皮细胞（HUVEC）和PMEC相比，HEVEC中NF-HEV的表达较强。原位杂交和免疫组化显示，NF-HEV的mRNA和蛋白质的表达在高水平，而选择性的HEVEC在人类扁桃体，派尔集合淋巴结，和淋巴结。发现NF-HEV蛋白含有二分核定位信号，并且当在HUVECs和HeLa细胞中异位表达时靶向于核。此外，内源性NF-HEV被发现在原位局限于扁桃体HEVECs的核。最后，线程和分子模拟研究表明，NF-HEV的氨基末端部分（aa 1-60）对应于一个新的同源结构域样螺旋-转角-受阻（HTH）DNA结合结构域。与在诱导淋巴管内皮细胞表型中起关键作用的非典型同源结构域转录因子Prox-1类似，NF-HEV可能是控制专门的HEV表型的关键核因子之一。
Lymphocyte homing to secondary lymphoid tissue and lesions of chronic inflammation is directed by multi-step interactions between the circulating cells and the specialized endothelium of high endothelial venules (HEVs). in this study, we used the PCR-based method of suppression subtractive hybridization (SSH) to identify novel HEV genes by comparing freshly purified HEV endothelial cells (HEVECs) with nasal polyp-derived microvascular endothelial cells (PMECs). By this approach, we cloned the first nuclear factor preferentially expressed in HEVECs, designated nuclear factor from HEVs (NF-HEV). Virtual Northern and Western blot analyses showed strong expression of NF-HEV in HEVECs, compared to human umbilical vein endothelial cells (HUVECs) and PMECs. In situ hybridization and immunohistochemistry revealed that NF-HEV mRNA and protein are expressed at high levels and rather selectively by HEVECs in human tonsils, Peyers's patches, and lymph nodes. The NF-HEV protein was found to contain a bipartite nuclear localization signal, and was targeted to the nucleus when ectopically expressed in HUVECs and HeLa cells. Furthermore, endogenous NF-HEV was found in situ to be confined to the nucleus of tonsillar HEVECs. Finally, threading and molecular modeling studies suggested that the amino-terminal part of NF-HEV (aa 1-60) corresponds; to a novel homeodomain-like Helix-Turn-Helix (HTH) DNA-binding domain. Similarly to the atypical homeodomain transcription factor Prox-1, which plays a critical role in the induction of the lymphatic endothelium phenotype, NF-HEV may be one of the key nuclear factors that controls the specialized HEV phenotype.