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

盘状结构域受体1(DDR1)酪氨酸激酶是一个细胞表面受体家族，它与几种类型的胶原结合，促进细胞黏附，已知与多种癌症有关。然而，以前还没有研究探讨DDR1在垂体腺瘤中的表达和功能。组织芯片分析52例垂体腺瘤组织中DDR1mRNA的表达水平表明，DDR1mRNA的表达与激素背景显著相关(Kruskal-Wallis检验；p&lt；0.0001)。为了进一步阐明DDR1在垂体腺瘤中的作用，我们分别利用DDR1克隆转染法和基于siRNA的DDR1基因沉默技术，在人垂体腺瘤细胞系HP-75中建立了DDR1高表达和低表达细胞系。实时定量RT-PCR和Western blotting证实，DDR1亚型(DDR1a和DDR1b)的表达在克隆转染组上调，而在siRNA组表达减弱。Matrigel侵袭实验显示，高表达DDR1的HP-75细胞侵袭能力增强，低表达DDR1的细胞侵袭能力降低。与此相一致的是，酶谱显示，在DDR1高表达和低表达的细胞中，基质金属蛋白酶-2和-9的激活水平分别增加和降低。体外检测细胞与I型、II型、III型和IV型胶原黏附对基质金属蛋白酶-2和-9表达的影响显示，DDR1调节细胞对I型胶原的黏附，这是导致DDR1高表达细胞分泌基质金属蛋白酶-2和-9的原因。综上所述，这些结果有力地表明，DDR1在垂体腺瘤细胞中介导了I型胶原与基质金属蛋白酶-2和-9之间的细胞侵袭相关信号。

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.