Characterization of VLA-3 integrin-mediated adhesion and its expre ision in cancer cells

VLA-3整合素介导的粘附的表征及其在癌细胞中的表达

• 批准号: 13672308
• 负责人: TSUJI Tsutomu
• 金额: $ 2.24万
• 依托单位: Hoshi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672308/
• 关键词: Integrin; VLA-3; Cell adhesion; Laminin; Cell motility; Transcription factor; Luciferase assay; Promoter; 転写制御; ルシフェラーゼ; 細胞接着分子; Ets; ラミニン5; 上皮細胞; メラノーマ

Cell adhesion molecules play a crucial role in the initial process of cancer metastasis through the cell-matrix interaction. In this study, we investigated the role of VLA-3 (α3β1 integrin) in invasion and metastasis of cancer cells and the regulation mechanism of its expression.1. Role of VLA-3 integrin in the motility of melanoma cells: We evaluated the role of soluble factors produced by epidermal cells in A375 melanoma cell motility by using the Boyden chamber chemoinvasion system. The motility-stimulating activity was associated with the α3 integrin-dependent adhesion-promoting activity and identified as laminin-5. Purified laminin-5 was capable of potentiating melanoma cell migration as measured in either the chemotaxis assay or the haptotaxis assay. Furthermore, immobilized laminin-5 induced A375 melanoma cells to secrete matrix metalloproteinase-9 (type IV collagenase) into the culture medium. These results strongly suggest that the interaction of laminin-5 produced in the epidermis with the α3β1 integrin on melanoma cells is involved in cell migration, invasion, and degradation of extracellular matrix proteins.2. Regulatory mechanism of the α3 integrin expression: We have cloned an approximately 4 kb DNA fragment of the 5'-flanking region of the murine α3 integrin gene and analyzed its promoter activity. Transfection of MKN1 gastric carcinoma cells with serially truncated segments of this region linked to a luciferase gene indicated that the sequence between positions -260 and -119 bp is required for efficient transcription in the cells. The sequence analysis of this segment showed the presence of several consensus sequences for transcription factors. The introduction of mutation in one of the Ets-binding sequences greatly decreased its promoter activity, suggesting that the transcription of the α3 integrin gene in these cells is regulated by the Ets-family of transcription factors.

细胞黏附分子通过细胞与基质的相互作用在肿瘤转移的初始过程中起着至关重要的作用。在本研究中，我们研究了Vla-3(α3β1整合素)在癌细胞侵袭转移中的作用及其表达调控机制。VLA-3整合素在黑色素瘤细胞运动中的作用：我们利用Boyden小室化学侵袭系统评价了表皮细胞产生的可溶性因子在A375黑色素瘤细胞运动中的作用。这种运动刺激活性与α3整合素依赖的黏附促进活性有关，鉴定为层粘连蛋白-5。纯化的层粘连蛋白-5能够增强黑色素瘤细胞的迁移，无论是在趋化试验中还是在触碰试验中都是如此。此外，固定化层粘连蛋白-5诱导A375黑色素瘤细胞分泌基质金属蛋白酶-9(IV型胶原酶)到培养基中。这些结果有力地表明，表皮产生的层粘连蛋白-5与黑色素瘤细胞上的α3β1整合素的相互作用参与了细胞的迁移、侵袭和细胞外基质蛋白的降解。α3整合素表达调控机制：我们克隆了小鼠α3整合素基因5‘侧翼区约4kb的基因片段，并对其启动子活性进行了分析。用与荧光素酶基因相连的该区域的连续截断片段转染MKN1胃癌细胞表明，在-260和-119bp之间的序列是细胞有效转录所必需的。该片段的序列分析表明，存在几个共同的转录因子序列。其中一个Ets结合序列的突变大大降低了其启动子活性，表明α3整合素基因在这些细胞中的转录受Ets转录因子家族的调控。

项目成果

Ogawa Y, Iwama M, Ohgi K, Tsuji T, Irie M, Itagaki T, Kobayashi H, Inokuchi N.: "Effect of replacing the aspartic acid/glutamic acid residues of bullfrog sialic acid binding lectin with asparagine/glutamine and arginine on the inhibition of cell prolifera

Okawa Y、Iwama M、Ohgi K、Tsuji T、Irie M、Itagaki T、Kobayashi H、Inokuchi N.：“用天冬酰胺/谷氨酰胺和精氨酸取代牛蛙唾液酸结合凝集素的天冬氨酸/谷氨酸残基对

Kato, T., Katabami, K., et al.: "Characterization of the promoter for the mouse α3 integrin gene. Involvement of the Ets-family of transcription factors in the promoter activity"European Journal of Biochemistry. 269巻. 4524-4532 (2002)

Kato, T.、Katabami, K. 等人：“小鼠 α3 整联蛋白基因启动子的表征。转录因子 Ets 家族参与启动子活性”《欧洲生物化学杂志》269. 4524。 - 4532 (2002)

Kato T, Katabami K, Takatsuki H, Han SA, Takeuchi K, Irimura T, Tsuji T.: "Characterization of the promoter for the mouse α3 integrin gene. Involvement of the Ets-family of transcription factors in the activity of α3 injtegrin gene promoter."Eur. J. Bioch

Kato T、Katabami K、Takatsuki H、Han SA、Takeuchi K、Irimura T、Tsuji T.：“小鼠 α3 整合素基因启动子的表征。转录因子 Ets 家族参与 α3 整合素基因的活性启动子。”Eur. J. Bioch

Kato T, Katabami K: "Characterization of the promoter for the mouse α3 integrin gene"Eur.J.Biochem.. 269. 4524-4532 (2002)

Kato T，Katabami K：“小鼠 α3 整联蛋白基因启动子的表征”Eur.J.Biochem.. 269. 4524-4532 (2002)

Tsuji T, Kawada Y, Kai-Murozono M, Komatsu S, Han SA, Takeuchi K, Mizushima H, Miyazaki, K, Irimura T.: "Regulation of melanoma cellmigration and invasion by laminin-5 and α3β1 integrin (VLA-3)."Clin. Exp. Metastasis. 19. 127-134 (2002)

Tsuji T，Kawada Y，Kai-Murozono M，Komatsu S，Han SA，Takeuchi K，Mizushima H，Miyazaki，K，Irimura T.：“层粘连蛋白-5和α3β1整合素（VLA-3）对黑色素瘤细胞迁移和侵袭的调节) .“临床实验转移。19. 127-134 (2002)