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Molecular aspect of in cell invasion by pituitary adenoma mediated by hypoxia-relating transcription factor

缺氧相关转录因子介导的垂体腺瘤细胞侵袭的分子方面

基本信息

批准号：
17591536
负责人：
YOSHIDA Daizo
金额：
$ 2.41万
依托单位：
Nippon Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17591536/
关键词：
cell invasion discoidin-domain receotor 1 hypoxia Matrix metallonroteinase Pituitary adenoma zymography 蛍光免疫染色 SDF-1 ELISA 浸潤 MMP RECK cDNA microarray siRNA 3-D invasion assay リン酸化 CREB

项目摘要

The discoidin domain receptor 1 (DDR1) tyrosine kinases are a family of cell surface receptors that bind to several types of collagen and facilitate cell adhesion that is known association with several cancers. However, no previous study has examined the expression and function of DDR1 in pituitary adenoma. Tissue microarray analysis of DDR1 expression levels in 52 pituitary adenoma tissues revealed that DDR1 expression was significantly related to hormonal background (Kruskal-Wallis test ; p<0.0001). To further elucidate the function of DDR1 in pituitary adenoma, we developed DDR1 over- and under-expressing cell lines using DDR1 clone transfection and siRNA-based DDR1 gene silencing, respectively, in a human pituitary adenoma cell line (HP-75). Real-time RT-PCR and Western blotting confirmed that expression of both DDR1 isoforms (DDRla and DDR1b) was elevated by clone transfection and diminished by siRNA. Matrigel invasion assays revealed that cell invasion was increased in HP-75 cells over-expressing DDR1 and decreased in cells under-expressing DDR1. Consistent with this, zymography revealed that the activation levels of matrix metalloproteinase (MMP)-2 and -9 were increased and decreased in cells over- and under-expressing DDR1, respectively. Examination of in vitro cell adhesion to collagen types I, II, III and IV with respect to MMP-2 and -9 expression revealed that DDR1 regulated cell adhesion to collagen type I, which was responsible for accelerating secretion of MMP-2 and -9 in DDR1 over-expressing cells. Taken together, these results strongly suggest that DDR1 mediates cell invasion-related signaling between collagen type I and MMP-2 and -9 in pituitary adenoma cells.

盘状蛋白结构域受体1 （DDR1）酪氨酸激酶是一个细胞表面受体家族，与几种类型的胶原蛋白结合，促进细胞粘附，已知与几种癌症有关。然而，目前尚无研究对DDR1在垂体腺瘤中的表达和功能进行研究。组织芯片分析52个垂体腺瘤组织中DDR1表达水平显示，DDR1表达与激素背景显著相关（Kruskal-Wallis检验，p<0.0001）。为了进一步阐明DDR1在垂体腺瘤中的功能，我们在人垂体腺瘤细胞系（HP-75）中分别使用DDR1克隆转染和基于sirna的DDR1基因沉默技术，建立了DDR1过表达和低表达细胞系。Real-time RT-PCR和Western blotting证实，克隆转染后DDR1亚型（DDRla和DDR1b）表达升高，siRNA转染后表达降低。侵袭实验显示，过表达DDR1的HP-75细胞侵袭增加，低表达DDR1的HP-75细胞侵袭减少。与此一致的是，酶谱分析显示，在DDR1过表达和过表达的细胞中，基质金属蛋白酶(MMP)-2和-9的激活水平分别升高和降低。检测细胞对I型、II型、III型和IV型胶原黏着的MMP-2和-9的表达，发现DDR1调节细胞对I型胶原的黏着，这是DDR1过表达细胞加速MMP-2和-9分泌的原因。综上所述，这些结果强烈提示DDR1介导垂体腺瘤细胞中I型胶原与MMP-2和-9之间的细胞侵袭相关信号传导。