Nakada Mitsutoshi, Miyamori Hisashi, Yamashita Junkoh, Sato Hiroshi
Department of Molecular Virology and Oncology, Kanazawa University, Ishikawa 920-0934, Japan.
Cancer Res. 2003 Jun 15;63(12):3364-9.
Testican family proteins are putative extracellular heparan/chondroitin sulfate proteoglycans of unknown function. We identified recently N-Tes, which is a product of testican 3 splicing variant gene, as an inhibitor of membrane-type matrix metalloproteinases (MT-MMPs). The inhibitory function is common among testican family members except for testican 2, which was shown to uniquely abolish inhibition of MT1-MMP- or MT3-MMP-mediated pro-MMP-2 activation by other testican family members. Testican 2 inactivates N-Tes by binding to the COOH-terminal extracellular calcium-binding domain of N-Tes through its NH(2)-terminal unique domain as demonstrated by coimmunoprecipitation analysis, and, thus, testican 2 was unable to inactivate a N-Tes deletion mutant lacking the extracellular calcium-binding domain (N-Tes-Delta 122). Migration of U251 cells on collagen, which was dependent on MT1-MMP activity under serum-free condition, was inhibited by N-Tes or N-Tes-Delta 122 deposited on collagen. Testican 2 was not incorporated into collagen by itself, and was deposited only in the presence of N-Tes, suggesting that testican 2 binds to N-Tes deposited on collagen. Binding of testican 2 to N-Tes deposited on collagen allowed migration of cells expressing MT1-MMP. Unlike wild-type N-Tes, N-Tes-Delta 122 did not bind to testican 2, and, thus, expression of testican 2 did not recover cell migration blocked by N-Tes-Delta 122. In situ hybridization showed that neurons are a major source of all of the testican family members in the normal brain. The quantitative reverse transcription-PCR analysis demonstrated that all of the testican family members are expressed prominently in normal brain, and their expression levels decrease as tumor grade increases. The expression level of testican 2 was the highest among testican family members regardless of histological grade of astrocytic tumors. These results suggest that abundant distribution of testican 2 may contribute to glioma invasion by inactivating other testican family members including N-Tes, which all inhibit MT-MMPs. We propose that N-Tes-Delta 122, which is resistant to testican 2, may have therapeutic potential as a barrier against glioma invasion.
睾丸蛋白聚糖家族蛋白是功能未知的假定细胞外硫酸乙酰肝素/硫酸软骨素蛋白聚糖。我们最近鉴定出N-Tes，它是睾丸蛋白聚糖3剪接变体基因的产物，是膜型基质金属蛋白酶（MT-MMPs）的抑制剂。除睾丸蛋白聚糖2外，这种抑制功能在睾丸蛋白聚糖家族成员中普遍存在，睾丸蛋白聚糖2已被证明能独特地消除其他睾丸蛋白聚糖家族成员对MT1-MMP或MT3-MMP介导的前MMP-2激活的抑制作用。免疫共沉淀分析表明，睾丸蛋白聚糖2通过其NH(2)-末端独特结构域与N-Tes的COOH-末端细胞外钙结合结构域结合，从而使N-Tes失活，因此，睾丸蛋白聚糖2无法使缺乏细胞外钙结合结构域的N-Tes缺失突变体（N-Tes-Delta 122）失活。在无血清条件下，U251细胞在胶原上的迁移依赖于MT1-MMP活性，而沉积在胶原上的N-Tes或N-Tes-Delta 122可抑制这种迁移。睾丸蛋白聚糖2本身不会整合到胶原中，只有在N-Tes存在时才会沉积，这表明睾丸蛋白聚糖2与沉积在胶原上的N-Tes结合。睾丸蛋白聚糖2与沉积在胶原上的N-Tes结合可使表达MT1-MMP的细胞迁移。与野生型N-Tes不同，N-Tes-Delta 122不与睾丸蛋白聚糖2结合，因此，睾丸蛋白聚糖2的表达不能恢复被N-Tes-Delta 122阻断的细胞迁移。原位杂交显示，神经元是正常大脑中所有睾丸蛋白聚糖家族成员的主要来源。定量逆转录-PCR分析表明，所有睾丸蛋白聚糖家族成员在正常大脑中均有显著表达，且其表达水平随肿瘤分级增加而降低。无论星形细胞瘤的组织学分级如何，睾丸蛋白聚糖2的表达水平在睾丸蛋白聚糖家族成员中都是最高的。这些结果表明，睾丸蛋白聚糖2的大量分布可能通过使包括N-Tes在内的其他抑制MT-MMPs的睾丸蛋白聚糖家族成员失活，从而促进胶质瘤的侵袭。我们提出，对睾丸蛋白聚糖2具有抗性的N-Tes-Delta 122可能具有作为抗胶质瘤侵袭屏障的治疗潜力。
