Tournier J M, Polette M, Hinnrasky J, Beck J, Werb Z, Basbaum C
Department of Anatomy, University of California, San Francisco 94143-0452.
J Biol Chem. 1994 Oct 14;269(41):25454-64.
Tracheal gland morphogenesis and gland hypertrophy in disease involve the penetration of epithelial cells into the submucosa, a process that requires digestion of the basal lamina and the surrounding extracellular matrix. We observed that bovine tracheal gland cells invaded collagen substrates and were inhibited from doing so in the presence of a metalloproteinase inhibitor GM6001. The gland cells, but not bovine tracheal surface epithelial cells, secreted a 72-kDa metalloproteinase. The purified enzyme could be activated with 4-aminophenylmercuric acetate and converted to an active 65-kDa form that was far more effective in degrading denatured collagen (gelatin) than nondenatured type I and IV collagens and was ineffective in degrading intact interstitial collagen fibers. At 25 degrees C, the initial rate of degradation of acid-solubilized type I collagen was approximately 50 mg of type I collagen cleaved per min per mg of enzyme, whereas acid-solubilized type IV collagen was degraded at approximately 250 mg cleaved per min per mg of enzyme. In contrast, at the same temperature, heat-denatured type I collagen was degraded 1000-fold more rapidly, while heat-denatured type IV collagen was cleaved 50-fold more rapidly. The activity of the enzyme was maximal at pH 7-8 and was completely abolished by the metalloproteinase inhibitors EDTA and 1,10-phenanthroline. In immunoblots, the enzyme was recognized by an antibody directed against human gelatinase A, the 72-kDa gelatinase. The purified enzyme disrupted the distribution pattern of type IV collagen in the gland basal lamina, as well as of interstitial collagen in the underlying stromal tissue, as shown in tissue sections by immunocytochemistry. Using an antibody directed against the purified enzyme, we also showed by immunocytochemistry that the gelatinase was present in tracheal tissue and was specifically located at the periphery of some tracheal gland acini. Northern blots showed higher concentrations of gelatinase A mRNA in glands than in epithelium microdissected from adult cow tracheas. These data indicate that gelatinase A is a specialized product of the tracheal gland epithelial cell, a cell type normally invasive as part of its developmental program; the enzyme may play an important role in normal gland development and disease-associated hypertrophy.
气管腺的形态发生以及疾病中的腺体肥大涉及上皮细胞向黏膜下层的浸润，这一过程需要消化基底膜和周围的细胞外基质。我们观察到牛气管腺细胞能够侵入胶原底物，而在金属蛋白酶抑制剂GM6001存在的情况下这种侵入会受到抑制。腺细胞，而非牛气管表面上皮细胞，分泌一种72-kDa的金属蛋白酶。纯化后的酶可用对乙酰氨基苯汞激活，并转化为有活性的65-kDa形式，该形式在降解变性胶原（明胶）方面比天然的I型和IV型胶原更有效，而在降解完整的间质胶原纤维方面无效。在25℃时，酸溶性I型胶原的初始降解速率约为每分钟每毫克酶可裂解50毫克I型胶原，而酸溶性IV型胶原的降解速率约为每分钟每毫克酶可裂解250毫克。相比之下，在相同温度下，热变性I型胶原的降解速度快10阗倍，而热变性IV型胶原的裂解速度快50倍。该酶的活性在pH 7-8时最大，并且会被金属蛋白酶抑制剂EDTA和1,10-菲咯啉完全抑制。在免疫印迹中，该酶可被一种针对人明胶酶A（72-kDa明胶酶）的抗体识别。如免疫细胞化学在组织切片中所示，纯化后的酶破坏了腺体基底膜中IV型胶原以及下方基质组织中间质胶原的分布模式。使用一种针对纯化酶的抗体，我们还通过免疫细胞化学表明，明胶酶存在于气管组织中，并且特异性地位于一些气管腺泡的周边。Northern印迹显示，腺体中明胶酶A mRNA的浓度高于从成年母牛气管显微切割得到的上皮中的浓度。这些数据表明，明胶酶A是气管腺上皮细胞的一种特殊产物，气管腺上皮细胞作为其发育程序的一部分通常具有侵袭性；该酶可能在正常腺体发育和疾病相关的肥大中起重要作用。
