The analysis of transcription factor and ECM degradation enzyme associated with invasion of glioblastoma

蛋白水解性细胞外基质(ECM)降解是胶质母细胞瘤侵袭的关键步骤。虽然多种蛋白水解酶参与了这一过程，但基质金属蛋白酶(MMPs)成员，尤其是基质金属蛋白酶-2，可能在降解过程中起着核心作用。为了发挥其酶活性，需要膜型MMPs(MT-MMPs)如MT1-MMPs对其进行蛋白水解性激活。在本研究中，我们通过表达克隆的方法筛选了MT1-基质金属蛋白酶介导的基质金属蛋白酶-2原加工调节因子的人胎儿c DNA文库，并分离到一个干扰原-基质金属蛋白酶-2活性的产物。它编码睾丸蛋白3的N-末端313个氨基酸，因此被命名为N-TES。睾丸蛋白1和3的表达而不是睾丸蛋白2的表达也抑制了MT1-MMPs对MMP2的激活。N-TES中氨基酸残基的缺失和替换表明，N-TES的N-末端110个氨基酸区域足以抑制MT1-MMPs激活的原-MMP2。此外，我们还展示了…此外，睾丸蛋白2通过其N端独特的结构域与N-TES的胞外钙结合(EC)结构域结合，从而使N-TES失活。U251细胞在胶原上的迁移依赖于基质金属蛋白酶的活性，并被沉积在胶原上的缺失EC结构域的N-TES或N-TES缺失突变体(N-TES-Δ122)所抑制。睾丸蛋白2与沉积在胶原上的N-TES结合，允许表达MT1-MMPs的细胞迁移。与N-TES不同，N-TES-Δ122不与睾丸蛋白2结合，因此睾丸蛋白2的表达不能恢复N-TES-Δ122阻断的细胞迁移。原位杂交显示，神经元是正常脑内所有睾丸蛋白家族成员的主要来源。定量逆转录聚合酶链式反应分析表明，睾丸蛋白原家族的所有成员在正常脑组织中均有表达，且随着肿瘤分级的增加，其表达水平逐渐降低。无论星形细胞肿瘤的组织学分级如何，睾丸蛋白2在睾丸蛋白家族成员中的表达水平最高。这些结果表明，N-TES和TECAN 1、3是脑内的ECM，通过抑制MT-MMPs而干扰肿瘤的侵袭，而TECAN 2的丰富分布可能通过抑制包括N-TES在内的其他TECN家族成员来抑制MT-MMPs而参与胶质瘤的侵袭。我们认为，N-TES-Δ122对睾丸蛋白2具有抵抗力，可能具有潜在的新功能，可以作为脑胶质瘤侵袭的屏障。较少

Proteolytic extracellular matrix (ECM) degradation is a key step in glioblastoma invasion. Although various proteinases are involved in the process, members of matrix metalloproteinases (MMPs), especially MMP-2, may play a central role in the degradation. To exert its enzymatic activity, pro-MMP-2 requires proteolytic activation by membrane-type MMPs (MT-MMPs) such as MT1-MMP. In the present study, we have screened a human fetal cDNA library by expression cloning for the regulator of pro-MMP-2 processing mediated by MT1-MMP and isolated a cDNA whose product interfered with pro-MMP-2 activation. It encodes N-terminal 313 amino acids regions of testican 3, and thus it was named N-Tes. Expression of testican 1 and testican 3 but not testican 2 also inhibited pro-MMP-2 activation by MT1-MMP. Deletion and substitution of amino acids residues in N-Tes revealed that N-terminal 110 amino acid region of N-Tes is enough for the inhibition of pro-MMP-2 activation by MT1-MMP. In addition, we showe … More d that testican 2 inactivates N-Tes by binding to the C-terminal extracellular calcium-binding (EC) domain of N-Tes through its N-terminal unique domain. Migration of U251 cells on collagen was dependent on MT1-MMP activity and was inhibited by N-Tes or N-Tes deletion mutant lacking the EC domain (N-Tes-Δ122) deposited on collagen. Binding of testican 2 to N-Tes deposited on collagen allowed migration of cells expressing MT1-MMP. Unlike N-Tes, N-Tes-Δ122 did not bind to testican 2, and thus expression of testican 2 did not recover cell migration blocked by N-Tes-Δ122. In situ hybridization showed that neurons are major source of all testican family members in the normal brain. The quantitative reverse transcription-polyraerase chain reaction analysis demonstrated that all members of testican family are expressed predominantly 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 N-Tes and testican 1, 3, which are brain ECM, interfere with tumor invasion by inhibiting MT-MMPs and 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-Δ122, which is resistant to testican 2, may have potential novel function as a barrier against glioma invasion. Less