Mackay A R, Gomez D E, Nason A M, Thorgeirsson U P
Office of the Director, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
Lab Invest. 1994 Jun;70(6):800-6.
Based on a previous observation of laminin-mediated increase in type IV collagenolytic activity of human melanoma (A-2058) and fibrosarcoma (HT-1080) cell lines (Turpeenniemi-Hujanen T, et al. Laminin increases the release of type IV collagenase from malignant cells. J Biol Chem 1986;261:1883-1889), the goal of this study was to identify the proteinases involved.
A soluble type IV collagenase assay and substrate gel electrophoresis were used to assess the effect of laminin and laminin peptides on type IV collagenolytic activity.
The laminin-mediated increase in type IV collagenolytic activity was not due to augmented expression or induction of three known type IV collagenolytic matrix metalloproteinases (MMP), i.e., 72-kilodalton gelatinase/type IV collagenase (MMP-2), stromelysin (MMP-3), and 92 kilodalton gelatinase/type IV collagenase (MMP-9). Furthermore, laminin did not modulate the expression of the tissue inhibitors of metalloproteinases (TIMP), TIMP-1 and TIMP-2. The E-8 fragment of laminin and the YIGSR laminin peptide had no effect on type IV collagenolytic or MMP/TIMP activities. However, the IKVAV containing PA22-2 laminin peptide selectively stimulated type IV collagenolytic activity of the A-2058 melanoma cell line, although it did not modulate MMP/TIMP activity. Laminin from three different sources of the Engelbreth-Holm-Swarm tumor was found to contain type IV collagenolytic activity. When laminin was added to harvested culture supernatants of the A-2058 and HT-1080 cell lines, the increase in type IV collagenolytic activity was comparable with that observed in supernatants from cells incubated with laminin for 48 hours. Analysis of the laminin preparations revealed five MMP forms ranging in molecular weight from approximately 58 to 105 kilodalton, which may represent latent and active forms of MMP-2 and MMP-9.
These findings suggest that proteinases present in the Engelbreth-Holm-Swarm laminin may account for most, if not all, of the observed laminin-mediated increase in type IV collagenolytic activity. However, the PA22-2-mediated increase in type IV collagenolytic activity of the A-2058 melanoma line remains to be elucidated.
基于先前观察到层粘连蛋白可介导人黑色素瘤（A - 2058）和纤维肉瘤（HT - 1080）细胞系IV型胶原酶活性增加（Turpeenniemi - Hujanen T等人，《层粘连蛋白增加恶性细胞中IV型胶原酶的释放》，《生物化学杂志》1986年；261:1883 - 1889），本研究的目的是确定其中涉及的蛋白酶。
采用可溶性IV型胶原酶测定法和底物凝胶电泳来评估层粘连蛋白和层粘连蛋白肽对IV型胶原酶活性的影响。
层粘连蛋白介导的IV型胶原酶活性增加并非由于三种已知的IV型胶原酶解基质金属蛋白酶（MMP）的表达增强或诱导，即72千道尔顿明胶酶/IV型胶原酶（MMP - 2）、基质溶解素（MMP - 3）和92千道尔顿明胶酶/IV型胶原酶（MMP - 9）。此外，层粘连蛋白并未调节金属蛋白酶组织抑制剂（TIMP）TIMP - 1和TIMP - 2的表达。层粘连蛋白的E - 8片段和YIGSR层粘连蛋白肽对IV型胶原酶解或MMP/TIMP活性无影响。然而，含有IKVAV的PA22 - 2层粘连蛋白肽选择性地刺激了A - 2058黑色素瘤细胞系的IV型胶原酶活性，尽管它并未调节MMP/TIMP活性。发现来自Engelbreth - Holm - Swarm肿瘤的三种不同来源的层粘连蛋白具有IV型胶原酶活性。当将层粘连蛋白添加到A - 2058和HT - 1080细胞系收获的培养上清液中时，IV型胶原酶活性的增加与在与层粘连蛋白孵育48小时的细胞上清液中观察到的增加相当。对层粘连蛋白制剂的分析揭示了五种分子量约为58至105千道尔顿的MMP形式，这可能代表MMP - 2和MMP - 9的潜在和活性形式。
这些发现表明，Engelbreth - Holm - Swarm层粘连蛋白中存在的蛋白酶可能是观察到的层粘连蛋白介导的IV型胶原酶活性增加的大部分（如果不是全部）原因。然而，PA22 - 2介导的A - 2058黑色素瘤系IV型胶原酶活性增加的机制仍有待阐明。
