Coexpression of integrin αvβ3 and matrix metalloproteinase-2 (MMP-2) coincides with MMP-2 activation:: Correlation with melanoma progression
Coexpression of integrin αvβ3 and matrix metalloproteinase-2 (MMP-2) coincides with MMP-2 activation:: Correlation with melanoma progression
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DOI：
10.1046/j.1523-1747.2000.00114.x
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发表时间：
2000-10-01
影响因子：
6.5
通讯作者：
van Muijen, GNP
中科院分区：
文献类型：
--
作者：
Hofmann, UB;Westphal, JR;van Muijen, GNP
肿瘤细胞侵袭和转移的形成依赖于粘附和蛋白水解机制。先前的研究表明，基质金属蛋白酶-2和整合素α（V）β（3）的表达与黑色素瘤进展相关。最近，基质金属蛋白酶-2与α（v）β（3）的直接结合涉及在侵袭性细胞的细胞表面上呈递活化的基质金属蛋白酶-2。在这项研究中，我们调查了这一点，使用高转移性，α（v）β（3）阴性黑色素瘤细胞。MV 3和BLM细胞系，它们的β（3）-转染的α（v）β（3）表达对应物，衍生自这些细胞系的异种移植物，以及包括黑素瘤进展的所有阶段的新鲜人皮肤黑素瘤损伤。分别采用逆转录-聚合酶链反应、免疫组化、蛋白质印迹和酶谱分析研究基质金属蛋白酶-2的表达和活化状态。基质金属蛋白酶-2蛋白在体外表达在α（v）β（3）阴性和α（v）β（3）阳性细胞系中相似。然而，显着的差异存在于非活性和活性基质金属蛋白酶-2的定位。可溶性活性基质金属蛋白酶-2仅在α（v）β（3）阴性细胞系的条件培养基中检测到，而在α（v）β（3）阳性细胞系中检测不到。相反，活性基质金属蛋白酶-2仅存在于表达α（v）β（3）的转染子的细胞表面上，对参与基质金属蛋白酶-2活化的其他组分的Western印迹分析显示，在β（3）转染子中，proMT 1-基质金属蛋白酶向活化形式的加工增强，而金属蛋白酶-2的组织抑制剂的分泌减少。在体内，功能活性基质金属蛋白酶-2的存在是显着更高的异种移植物来源于α（v）β（3）表达MV 3和BLM细胞系。在人类皮肤黑色素瘤病变中，通过免疫组织化学测定，在原位黑色素瘤中既不能检测到基质金属蛋白酶-2也不能检测到整合素α（v）β（3）。相反，在原发性黑色素瘤和转移性黑色素瘤中，基质金属蛋白酶-2阳性和α（v）β（3）阳性肿瘤细胞的数量明显增加。双染色实验和激光共聚焦显微镜显示，在晚期原发性黑色素瘤和转移性黑色素瘤中，共表达基质金属蛋白酶-2和α（v）β（3）的细胞百分比增加。此外，酶谱分析表明，功能活性基质金属蛋白酶-2经常出现在黑色素瘤转移。在这些病变中，可检测到高比例的基质金属蛋白酶-2和α（v）β（3）双阳性黑素瘤细胞。我们的研究表明，激活的基质金属蛋白酶-2的存在与α（v）β（3）的表达在体外和体内的人黑色素瘤细胞，也在新鲜的人黑色素瘤病变。这些发现强烈表明，协调表达的两个因素可能需要黑色素瘤细胞的侵袭和转移的形成。
Tumor cell invasion and metastasis formation depend on both adhesive and proteolytic mechanisms. Previous studies have shown that expression of matrix metalloproteinase-2 and integrin alpha(v)beta(3) correlate with melanoma progression. Recently, direct binding of matrix metalloproteinase-2 to alpha(v)beta(3) was implicated in presenting activated matrix metalloproteinase-2 on the cell surface of invasive cells. In this study we investigated this, using the highly metastatic, alpha(v)beta(3)-negative melanoma cell. lines MV3 and BLM, their beta(3)-transfected alpha(v)beta(3) expressing counterparts, xenografts derived from these cell lines, and fresh human cutaneous melanoma lesions comprising all stages of melanoma progression. Expression and activation status of matrix metalloproteinase-2 were studied by reverse transcription-polymerase chain reaction, immunohistochemistry, western blotting, and zymographic analysis, respectively. Matrix metalloproteinase-2 protein expression in vitro was similar in both alpha(v)beta(3)-negative and alpha(v)beta(3)-positive cell lines. Remarkable differences, however, exist in the localization of inactive and active matrix metalloproteinase-2. Soluble active matrix metalloproteinase-2 was detectable only in the conditioned medium of alpha(v)beta(3)-negative cell lines and undetectable in the alpha(v)beta(3)-positive cell lines. Conversely, active matrix metalloproteinase-2 was present exclusively on the cell surface of the alpha(v)beta(3) expressing transfectants, Western blot analysis of other components that are involved in matrix metalloproteinase-2 activation showed that processing of proMT1-matrix metalloproteinase to the activated form was enhanced in beta(3) transfectants, whereas secretion of tissue inhibitor of metalloproteinase-2 was decreased. In vivo, the presence of functionally active matrix metalloproteinase-2 was significantly higher in xenografts derived from the alpha(v)beta(3) expressing MV3 and BLM cell lines. In human cutaneous melanoma lesions, neither matrix metalloproteinase-2 nor integrin alpha(v)beta(3) is detectable in melanoma in situ as determined by immunohistochemistry. In contrast, the number of matrix metalloproteinase-2-positive and alpha(v)beta(3)-positive tumor cells was clearly increased in primary melanomas, and melanoma metastases. Double staining experiments and confocal laser microscopy demonstrated that the percentage of cells coexpressing matrix metalloproteinase-2 and alpha(v)beta(3) increased in advanced primary melanomas and melanoma metastases. In addition, zymography showed that functionally active matrix metalloproteinase-2 was frequently present in melanoma metastases. In these lesions a high proportion of matrix metalloproteinase-2- and alpha(v)beta(3)-double-positive melanoma cells were detectable. Our study demonstrates that the presence of activated matrix metalloproteinase-2 correlates with expression of alpha(v)beta(3) in human melanoma cells both in vitro and in vivo, and also in fresh human melanoma lesions. These findings strongly suggest that co-ordinated expression of both factors may be required for melanoma cell invasion and metastasis formation.