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.

来自层粘连蛋白α1和α2链羧基末端球状结构域（G结构域）的活性序列已通过在许多生物测定中筛选重叠的合成肽来鉴定（Nomizu等人，J.Biol.Chem.270：20583-20590，1995 ；Nomizu 等人，FEBS Lett.396：37-42，1996）。我们在胚胎第 13 天小鼠的颌下腺外植体中测试了这些肽的活性，以确定参与器官发育的功能位点。层粘连蛋白 α1 链肽 RKRLQVQLSIRT（残基 2719-2730，指定为 AG-73）显着抑制上皮分支形态发生。相比之下，其他细胞粘附层粘连蛋白 α1 链肽（包括 AASIKVAVSADR 和 NRWHSIYITRFG）未能抑制分支。 MG-73 是层粘连蛋白 α2 链 AG-73 的同源物，不会抑制分支。 α2链肽没有作用，这可能是由于胚胎第13天颌下腺中该层粘连蛋白链的水平较低。层粘连蛋白 α2 链特异性单克隆抗体与发育腺泡的基底膜发生强烈反应，但仅对胚胎第 13 天下颌下上皮基底膜产生弱染色。通过免疫荧光检测，在 AG-73 和对照乱序肽处理的外植体上皮细胞中，E-钙粘蛋白和 α6 整联蛋白的表达没有变化。相反，巢蛋白/巢蛋白的免疫染色显示，用AG-73处理3天的外植体具有不连续的基底膜。用对照肽处理3天的外植体显示出正常的基底膜。这些结果表明，含有层粘连蛋白α1链AG-73序列的区域对于胚胎早期颌下腺的发育至关重要。 AG-73 处理的外植体中不连续的基底膜可能表明该区域在基底膜组装中发挥着重要作用。