Expression and function of laminin isoforma during organogenesis

器官发生过程中层粘连蛋白亚型的表达和功能

基本信息

批准号：
08670033
负责人：
KADOYA Yuichi
金额：
$ 1.41万
依托单位：
KITASATO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Active sequences from the laminin alpha1 and alpha2 chain carboxyl-terminal globular domains (Gdomain)have been identified by screening overlapping synthetic peptides in a number of biological assays (Nomizu et al., J.Biol.Chem.270 : 20583-20590,1995 ; Nomizu et al., FEBS Lett.396 : 37-42,1996). We have tested the activity of these peptides in submandibular gland explants of embryonic day-13 mice to determine the functional sites involved in organ development. The laminin alpha1 chain peptide, RKRLQVQLSIRT (residues 2719-2730-and designated AG-73), significantly inhibited epithelial branching morphogenesis. In contrast, other cell adhesive laminin alpha1 chain peptides including the AASIKVAVSADR and NRWHSIYITRFG failed to inhibit the branching. MG-73, a homologue of AG-73 from the laminin alpha2 chain, did not inhibit the branching. The alpha2 chain peptide had no effect, which may be due to the low levels of this laminin chain in embryonic day 13 submandibular gland. Laminin alpha2 chain-specific monoclocal antibodies strongly reacted with the basement membranes of developed acini but only weakly stained embryonic day 13 submandibular epithelial basement membrane. The expression of E-cadherin and alpha6 integrin, as detected by immunofluorescence, were unchanged in both AG-73 and control scramble peptide treated epithelial cells of the explants. In contrast, immunostaining of nidogen/entactin showed that explants treated with AG-73 for 3 days had a discontinuous basement membrane. Explants treated for 3 days with control peptide showed a normal basement membrane. These results suggest that the region containing the AG-73 sequence of the laminin alpha1 chain is crucial for development of submandibular gland at early embryonic stages. The discontinuous basement membrane in AG-73-treated explants may indicate an important role for this region in basement membrane assembly.

通过在许多生物学测定中筛选重叠的合成肽（Nomizu等，J.Biol.Chem.270：20583-20583-20590,1995; nomizu et al。 1996年37-42）。我们已经测试了这些肽在13小鼠的胚胎下腺外植体中的活性，以确定器官发育所涉及的功能部位。层粘连蛋白α1链肽RKRLQVQLSIRT（残基2719-2730和指定的AG-73）显着抑制上皮分支形态发生。相反，其他细胞粘附层粘连蛋白α1链肽在内，包括Aasikvavsadr和NRWHSiyItrFG未能抑制分支。 MG-73是层粘连蛋白α2链的AG-73的同源物，没有抑制分支。 Alpha2链肽没有作用，这可能是由于该层粘蛋白链在胚胎第13天的下颌腺中的水平较低。层粘连蛋白α2链特异性的单网局抗体与发达的acini的基底膜强烈反应，但仅染色弱染色的胚胎第13天下皮上皮地下膜。通过免疫荧光检测到的E-钙粘着蛋白和α6整合素的表达在AG-73和对照争夺肽处理过的上皮细胞中均未变化。相比之下，尼多基/肠子素的免疫染色表明，用AG-73处理3天的外植物的地下室膜不连续。用对照肽处理3天的外植体显示正常的地下膜。这些结果表明，层粘连蛋白α1链的AG-73序列的区域对于早期胚胎阶段的下颌腺发展至关重要。 AG-73处理的外植体中的不连续的地下膜可能表明该区域在基底膜组装中的重要作用。