Li L, Eisen A Z, Sturman E, Seltzer J L
Division of Dermatology, Washington University School of Medicine, Box 8123, St. Louis, MO 63110, USA.
Biochim Biophys Acta. 1998 Oct 21;1405(1-3):110-20. doi: 10.1016/s0167-4889(98)00091-3.
Fibroblasts in monolayer culture secrete gelatinase A (MMP2; 72 kDa type IV collagenase) only in its proenzyme form. Unlike other secreted matrix metalloproteinases, progelatinase A is refractory to activation by serine proteinases. Disparate agents, including monensin, cytochalasin D, and concanavalin A, have been found to mediate the activation of gelatinase A zymogen secreted by fibroblast monolayers. Our finding that monensin-mediated activation can be reversed by the protein tyrosine kinase inhibitor genistein (Li et al., Experimental Cell Research 232 (1997) 332) prompted us to investigate the effect of the specific inhibitor of protein tyrosine phosphatases, sodium orthovanadate, on progelatinase A activation. Treatment of fibroblast monolayers with orthovanadate also results in the secretion of activated gelatinase A. This activation is dose- and time-dependent, requires protein synthesis, and is associated with cell membranes. Vanadate-mediated activation does not occur in the presence of herbimycin A, a protein tyrosine kinase inhibitor. As with progelatinase activation mediated by monensin, concanavalin A, and cytochalasin D, orthovanadate treatment results in increased synthesis of the membrane proteinase MT1-MMP, that can catalyze the activation of progelatinase A. Protein tyrosine kinase inhibitors are able to prevent the increase of MT1-MMP mRNA, as shown by Northern blot and RT-PCR. In addition, orthovanadate potentiates the effects of monensin and concanavalin A. While treatment with monensin or concanavalin A result only in an increase of the putative activator MT1-MMP, orthovanadate also reduces the production of the specific inhibitor TIMP-2. These experiments implicate protein tyrosine phosphorylation in the signal transduction pathways which lead to the activation of progelatinase A.
单层培养的成纤维细胞仅以酶原形式分泌明胶酶A（MMP2；72 kDa IV型胶原酶）。与其他分泌型基质金属蛋白酶不同，前明胶酶A对丝氨酸蛋白酶的激活具有抗性。已发现包括莫能菌素、细胞松弛素D和伴刀豆球蛋白A在内的不同试剂可介导成纤维细胞单层分泌的明胶酶A酶原的激活。我们发现莫能菌素介导的激活可被蛋白酪氨酸激酶抑制剂染料木黄酮逆转（Li等人，《实验细胞研究》232 (1997) 332），这促使我们研究蛋白酪氨酸磷酸酶的特异性抑制剂原钒酸钠对前明胶酶A激活的影响。用原钒酸钠处理成纤维细胞单层也会导致活性明胶酶A的分泌。这种激活是剂量和时间依赖性的，需要蛋白质合成，并且与细胞膜相关。在蛋白酪氨酸激酶抑制剂赫伯霉素A存在的情况下，钒酸盐介导的激活不会发生。与莫能菌素、伴刀豆球蛋白A和细胞松弛素D介导的前明胶酶激活一样，原钒酸钠处理会导致膜蛋白酶MT1-MMP的合成增加，MT1-MMP可催化前明胶酶A的激活。如Northern印迹和RT-PCR所示，蛋白酪氨酸激酶抑制剂能够阻止MT1-MMP mRNA的增加。此外，原钒酸钠增强了莫能菌素和伴刀豆球蛋白A的作用。虽然用莫能菌素或伴刀豆球蛋白A处理只会导致假定激活剂MT1-MMP的增加，但原钒酸钠也会减少特异性抑制剂TIMP-2的产生。这些实验表明蛋白酪氨酸磷酸化参与了导致前明胶酶A激活的信号转导途径。
