Dog mastocytoma cells secrete a 92-kD gelatinase activated extracellularly by mast cell chymase
Dog mastocytoma cells secrete a 92-kD gelatinase activated extracellularly by mast cell chymase
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DOI：
10.1172/jci118583
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发表时间：
1996-04-01
影响因子：
15.9
通讯作者：
Caughey, GH
中科院分区：
文献类型：
--
作者：
Fang, KC;Raymond, WW;Caughey, GH
与结缔组织重塑和肿瘤侵袭有关的明胶溶解金属蛋白酶以无活性酶原的形式从几种类型的细胞中分泌，在本报告中，狗肥大细胞瘤细胞 BR 系分泌的明胶酶活性特征揭示了佛波醇诱导型，类似于 92-kD，Ca2+- 和 Zn2+ 依赖性酶原，随着时间的推移裂解为较小的活性形式，细胞孵化与一般丝氨酸蛋白酶抑制剂 PMSF 一起使用，可防止酶原裂解并使其纯化不含活化产物，纯化的肥大细胞瘤原明胶酶的 NH2 末端 13 个氨基酸与人、小鼠和兔 92-kD 明胶酶（明胶酶 B；基质金属蛋白酶-9）的 NH2 末端 13 个氨基酸具有 50-67% 的同一性。使用离子载体 A23187 使肥大细胞瘤细胞脱颗粒大大加速了酶原裂解，表明分泌颗粒中存在的丝氨酸蛋白酶将原明胶酶水解为活性片段。为了鉴定激活的蛋白酶，将细胞与离子载体和一组选择性丝氨酸蛋白酶抑制剂、大豆胰蛋白酶抑制剂和琥珀酰-L-Ala-Ala-Pro-Phe-氯甲基酮可抑制肥大细胞食糜酶，防止原明胶酶活化。类胰蛋白酶和狗肥大细胞蛋白酶 (dMCP)-3 的抑制剂，即抑肽酶或双(5-脒基-2-苯并咪唑基)甲烷 (BABIM)，则不会。在使用高度纯化的酶的进一步实验中，肥大细胞瘤细胞食糜酶在没有其他酶或辅因子的情况下激活 92-kD 原明胶酶；然而，类胰蛋白酶和 dMCP-3 没有效果。这些数据表明，狗肥大细胞瘤细胞分泌与原明胶酶B相关的金属蛋白酶，该金属蛋白酶在细胞外被胞吐的糜酶直接激活，并首次证明了细胞通过共同分泌激活酶来激活其分泌的基质金属蛋白酶。在肥大细胞瘤中，该途径可能促进肿瘤侵袭周围组织，而在正常肥大细胞中，它可以在组织重塑和修复中发挥作用。
Gelatinolytic metalloproteinases implicated in connective tissue remodeling and tumor invasion are secreted from several types of cells in the form of inactive zymogens, In this report, characterization of gelatinase activity secreted by the BR line of dog mastocytoma cells reveals a phorbol-inducible, similar to 92-kD, Ca2+- and Zn2+-dependent proenzyme cleaved over time to smaller, active forms, Incubation of cells with the general serine protease inhibitor, PMSF, prevented proenzyme cleavage and permitted its purification free of activation products, The NH2-terminal 13 amino acids of the purified mastocytoma progelatinase are 50-67% identical to those of human, mouse, and rabbit 92-kD progelatinase (gelatinase B; matrix metalloproteinase-9). Degranulation of mastocytoma cells using ionophore A23187 greatly accelerated proenzyme cleavage, suggesting that a serine protease present in secretory granules hydrolyzed the progelatinase to active fragments, To identify the activating protease, cells were coincubated with ionophore and a panel of selective serine protease inhibitors, Soybean trypsin inhibitor and succinyl-L-Ala-Ala-Pro-Phe-chloromethylketone, which inhibit mast cell chymase, prevented progelatinase activation. Inhibitors of tryptase and dog mast cell protease (dMCP)-3, i.e., aprotinin or bis(5-amidino-2-benzimidazolyl) methane (BABIM), did not. In further experiments using highly purified enzymes, mastocytoma cell chymase activated 92-kD progelatinase in the absence of other enzymes or cofactors; tryptase and dMCP-3, however, had no effect. These data demonstrate that dog mastocytoma cells secrete a metalloproteinase related to progelatinase B that is directly activated outside of the cell by exocytosed chymase, and provide the first demonstration of a cell that activates a matrix metalloproteinase it secretes by cosecreting an activating enzyme, In mastocytomas, this pathway may facilitate tumor invasion of surrounding tissues, and in normal mast cells, it could play a role in tissue remodeling and repair.