Li Zichen, Takino Takahisa, Endo Yoshio, Sato Hiroshi
Department of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan.
Central Research Resource Branch, Cancer Research Institute, Kanazawa University, Kanazawa, Japan.
Cancer Sci. 2017 Mar;108(3):347-353. doi: 10.1111/cas.13134. Epub 2017 Mar 16.
An artificial receptor for proMMP-9 was created by fusing tissue inhibitor of MMP-1 (TIMP-1) with type II transmembrane mosaic serine protease (MSP-T1). Expression of MSP-T1 in 293T cells induced binding of proMMP-9, which was processed by MMP-2 activated by membrane type 1 MMP (MT1-MMP). HT1080 cells transfected with the MSP-T1 gene produced activated MMP-9 in collagen gel, and addition of proMMP-2 to the culture augmented it, which resulted in intensive collagen digestion. These cells metastasized into chick embryonic liver more than control cells. Treatment of HT1080 cells with concanavalin A in the presence of exogenous proMMP-2 induced activation of not only proMMP-2 but also proMMP-9. Knockdown of MT1-MMP or TIMP-2 expression with siRNA suppressed activation of both proMMP-2 and proMMP-9. Transfection of TIMP-1 siRNA suppressed cell binding and activation of proMMP-9, but not proMMP-2 activation. Knockdown of a disintegrin and metalloproteinase 10 (ADAM10) expression reduced cell binding and processing of proMMP-9. These results suggest that proMMP-9, which binds to a receptor complex containing TIMP-1 and ADAM10, is activated by the MT1-MMP/MMP-2 axis, and MMP-9 thus activated stimulates cellular proteolysis and metastasis.
通过将基质金属蛋白酶-1组织抑制剂（TIMP-1）与II型跨膜镶嵌丝氨酸蛋白酶（MSP-T1）融合，构建了一种前基质金属蛋白酶-9（proMMP-9）的人工受体。MSP-T1在293T细胞中的表达诱导了proMMP-9的结合，proMMP-9由膜型1基质金属蛋白酶（MT1-MMP）激活的基质金属蛋白酶-2（MMP-2）进行加工处理。用MSP-T1基因转染的HT1080细胞在胶原凝胶中产生了活化的MMP-9，向培养物中添加proMMP-2可增强这种作用，进而导致强烈的胶原消化。这些细胞比对照细胞更易转移至鸡胚肝脏。在外源proMMP-2存在的情况下，用伴刀豆球蛋白A处理HT1080细胞不仅诱导了proMMP-2的活化，还诱导了proMMP-9的活化。用小干扰RNA（siRNA）敲低MT1-MMP或TIMP-2的表达可抑制proMMP-2和proMMP-9的活化。转染TIMP-1 siRNA可抑制细胞与proMMP-9的结合及活化，但不影响proMMP-2的活化。敲低解整合素金属蛋白酶10（ADAM10）的表达可减少细胞与proMMP-9的结合及加工处理。这些结果表明，与包含TIMP-1和ADAM10的受体复合物结合的proMMP-9被MT1-MMP/MMP-2轴激活，如此活化的MMP-9刺激细胞蛋白水解和转移。
