Intracellular activation of gelatinase A (72-kDa type IV collagenase) by normal fibroblasts.
Intracellular activation of gelatinase A (72-kDa type IV collagenase) by normal fibroblasts.
复制标题
正常成纤维细胞对明胶酶 A（72-kDa IV 型胶原酶）的细胞内激活。
DOI：
10.1073/pnas.94.9.4424
复制
发表时间：
1997
影响因子：
11.1
通讯作者：
Seltzer,JL
中科院分区：
文献类型：
--
作者：
Lee,AY;Akers,KT;Collier,M;Li,L;Eisen,AZ;Seltzer,JL
作为单层培养的正常成纤维细胞分泌基质金属蛋白酶（MMP），包括明胶酶A （72-kDa IV型胶原酶）作为失活酶原。先前我们发现，在I型胶原晶格中培养的正常成纤维细胞分泌活性明胶酶a。这里我们发现，明胶酶a的激活发生在细胞内，并且激活剂与高尔基膜结合。在胶原晶格中培养的成纤维细胞的细胞提取物在培养基中检测到活性酶之前至少4-6小时含有活性的62-kDa明胶酶A。脉冲追踪实验证实了这些结果。激活剂是膜结合的，定位于高尔基富集的部分。晶格培养的高纯度质膜不能将明胶酶A从酶原转化为活性形式。激活剂可能是一种金属蛋白酶，因为EDTA阻止外源前酶被膜组分激活。膜型MMP1被认为是负责激活肿瘤细胞质膜上的明胶酶A的酶，在晶格培养过程中，其mRNA和蛋白水平均未发生显著变化。细胞内明胶酶A mRNA和蛋白水平在培养期间升高，金属蛋白酶组织抑制剂浓度没有变化。由于无金属蛋白酶原酶的组织抑制剂作为激活物的底物的可用性更高，因此膜型MMP1可能是激活酶。在这种情况下，恶性转化可能涉及激活剂对质膜定位的改变。
Normal fibroblasts cultured as monolayers secrete matrix metalloproteinases (MMP), including gelatinase A (72-kDa type IV collagenase) as inactive zymogens. Previously we found that normal fibroblasts cultured in a type I collagen lattice (dermal equivalent) secrete active gelatinase A. Here we show that the activation of progelatinase A occurs within the cell and that the activator copurifies with Golgi membranes. Cell extracts of fibroblasts cultured in collagen lattices contain active 62-kDa gelatinase A at least 4–6 h before active enzyme is detected in the culture medium. Pulse–chase experiments confirm these results. The activator is membrane-bound and localizes to the Golgi-enriched fraction. Highly purified plasma membranes from lattice cultures are unable to convert gelatinase A from the zymogen to its active form. The activator may be a metalloproteinase because EDTA prevents activation of exogenous proenzyme by membrane fractions. Membrane-type MMP1, the enzyme thought to be responsible for activation of gelatinase A on the plasma membrane of tumor cells, shows no significant change in either mRNA or protein levels during lattice culture. Intracellular levels of gelatinase A mRNA and protein increase during the culture period, and tissue inhibitor of metalloproteinases concentration does not change. Because of the greater availability of tissue inhibitor of metalloproteinases-free proenzyme as a substrate for the activator, it is possible that membrane-type MMP1 is the activating enzyme. In that case, malignant transformation may involve a change in the localization of the activator to the plasma membrane.