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

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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