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

细胞外基质（ECM）的蛋白降解是胶质母细胞瘤侵袭的关键步骤。虽然多种蛋白酶参与了这一过程，但基质金属蛋白酶（MMP）的成员，特别是MMP-2，可能在降解过程中发挥核心作用。为了发挥其酶活性，pro-MMP-2需要通过膜型MMP（MT-MMP）如MT 1-MMP的蛋白水解活化。在本研究中，我们通过表达克隆的方法从人胎儿cDNA文库中筛选了MT 1-MMP介导的pro-MMP-2加工的调节因子，并分离了一个干扰pro-MMP-2活化的cDNA。它编码睾丸素3的N端313个氨基酸，因此命名为N-Tes。testican 1和testican 3的表达也抑制了MT 1-MMP对pro-MMP-2的激活，但testican 2的表达没有抑制。N-Tes氨基酸残基的缺失和替换表明，N-Tes的N端110个氨基酸区域足以抑制MT 1-MMP激活pro-MMP-2。此外，我们还展示了 ...更多信息 d睾丸蛋白聚糖2通过其N-末端独特结构域与N-Tes的C-末端细胞外钙结合（EC）结构域结合而使N-Tes失活。U251细胞在胶原上的迁移依赖于MT 1-MMP的活性，并可被N-Tes或缺失EC结构域的N-Tes缺失突变体（N-Tes-Δ122）抑制。睾丸素2与沉积在胶原上的N-Tes的结合允许表达MT 1-MMP的细胞迁移。与N-Tes不同，N-Tes-Δ122不与testisan 2结合，因此testisan 2的表达不能恢复被N-Tes-Δ122阻断的细胞迁移。原位杂交结果表明，神经元是正常脑内所有testican家族成员的主要来源。定量逆转录-聚合酶链反应（RT-PCR）分析表明，睾丸素家族的所有成员均在正常脑组织中表达，其表达水平随肿瘤分级的增高而降低。无论星形细胞肿瘤的组织学分级如何，testican 2的表达水平在testican家族成员中最高。这些结果表明，N-Tes和testican 1，3，这是脑ECM，干扰肿瘤的侵袭，通过抑制MT-MMPs和testican 2的丰富分布可能有助于胶质瘤的侵袭，通过灭活其他testican家族成员，包括N-Tes，这都抑制MT-MMPs。我们认为，对睾丸素2具有抗性的N-Tes-Δ122可能具有作为胶质瘤侵袭屏障的潜在新功能。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