Basement Membranes and Associated Protein Factors In Development and Disease

发育和疾病中的基底膜和相关蛋白质因子

• 批准号: 7593363
• 负责人: Yoshihiko Yamada
• 金额: $ 82.9万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593363
• 关键词: Address; Adult; Affect; Animal Model; Antibodies; Basement membrane; Binding; Binding Sites; Biological; Biological Process; Blood Vessels; Cartilage; Cell Adhesion; Cell Line; Cell Surface Receptors; Cell physiology; Cell-Matrix Junction; Cells; Collagen Type IV; Curare; Defect; Development; Disease; Disruption; Dysplasia; EWS/FLI 1 Type 1 antisense oligonucleotide; Electromyography; Embryo; Embryonic Development; Epithelial; Extracellular Matrix; Family; Fibroblasts; Genes; Glycoproteins; Goals; Heparin; Heparin Binding; Hereditary Disease; Homeostasis; Human; Individual; Integrin alpha2; Integrin alpha2beta1; Integrins; Knock-out; Knockout Mice; Laminin; Lung; Lymphoid Cell; Mediating; Membrane Proteins; Mesenchymal; Modeling; Morphogenesis; Mus; Muscle; Mutant Strains Mice; Mutation; Myocardial; Myotonia; NID gene; Neoplasm Metastasis; Neurons; Nidogen; Organ; Peptide Elongation Factor 1; Peptides; Perinatal; Phenotype; Play; Process; Protein Isoforms; Proteins; Recombinant Proteins; Recombinants; Role; Schwartz-Jampel Syndrome; Site; Skin; Structure; Structure-Activity Relationship; Tissues; Tumor Cell Invasion; Wound Healing; alpha Dystroglycan; angiogenesis; human disease; knockout gene; laminin alpha1; laminin-1; migration; mutant; neoplastic cell; perlecan; protein expression; scaffold; syndecan; tumor; tumor growth

Basement membranes are thin extracellular matrices that separate epithelial and mesenchymal cells and surround cells, such as endothelial, muscular, and neural cells. Basement membranes are the first extracellular matrix to appear in development and are critical for organ development and tissue repair. They not only provide the scaffold for cells and cell layers, but they also have an essential role in tissue morphogenesis that affects cell adhesion, migration, proliferation, and differentiation. Basement membranes provide major barriers in blood vessels to the passage of proteins and invasion by metastatic tumor cells. Basement membranes consist of collagen IV, laminin, perlecan, nidogen/entactin, and other molecules, which interact with each other to form the supramolecular structure and also bind cell surface receptors such as integrins and syndecans. Disruptions of these protein interactions and dysregulation of basement membrane protein expression cause impaired tissue development and diseases. Laminins are a family of large multidomain glycoproteins usually specific to basement membranes. To date, at least fifteen laminin isoforms, laminin-1 through laminin-15, have been identified. Laminin has a variety of biological activities, including promoting cell adhesion, migration, differentiation, tumor cell invasion, and interactions with matrix molecules and cell surface receptors. The LG4 module of laminin alpha1, a subunit of laminin-1, plays a critical role in early embryonic development. We previously identified active peptides AG73 and EF-1 from laminin alpha1 LG4 for binding to heparin/syndecan and integrin alpha2beta1, respectively. However, their activity and functional relationship within the laminin-1 protein and LG4 module are not known. To address this question, we prepared recombinant LG4 proteins that contain site-specific mutations within the AG73, EF-1, and alpha-dystroglycan binding sites and analyzed their binding activities to syndecans, integrins, and heparin and their activities for cell attachment and spreading and compared them with laminin-1. We found that recombinant proteins containing mutations within AG73 or the alpha-dystroglycan binding site lost heparin binding activity and did not attach to lymphoid cell lines expressing individual syndecans, suggesting that LG4 binds syndecans through these sites. These mutant LG4 proteins had significantly reduced fibroblast attachment, while mutant LG4 in EF-1 retained cell attachment activity but did not promote cell spreading. Fibroblast attachment to LG4 was inhibited by heparin but not by integrin antibodies, while cell spreading was inhibited by anti-integrin alpha2 but not by alpha6. In contrast, laminin-1-mediated fibroblast attachment and spreading were not inhibited by heparin or anti-integrin alpha2, indicating that LG4 and laminin-1 have distinct mechanisms for cell attachment and spreading. These results suggest that, similar to other laminin alpha chains, laminin alpha1 LG4-5 may also be produced by a proteolytic cleavage of the laminin alpha1 chain in certain tissues where it exerts its activity. Studies with gene knockout mice and human genetic diseases demonstrated that perlecan is essential for development and that the lack of perlecan results in either embryonic lethality due to defective myocardial basement membrane or perinatal lethality due to cartilage defects. In addition, perlecan is implicated in many biological functions in tissue homeostasis and diseases. We previously created perlecan knockout (Perl-/-) mice, which developed a severe chondrodysplasia and died as embryos or perinatally. We subsequently identified mutations of the perlecan genes in two human diseases, dyssegmental dysplasia, Silverman-Handmaker type (DDSH), characterized by lethal chondrodysplasia and Schwartz-Jampel syndrome (SJS), characterized by myotonia and milder chondrodysplasia. Animal models are useful to determine the roles of perlecan in adult tissue functions and diseases. However, the lethal phenotype of Perl-/- mice has hampered these studies. To overcome the problem, we created perinatal lethality-rescued Perl-/- mice by expressing recombinant perlecan specifically to cartilage but not other tissues. The mutant mice survived and developed myotonia, showing a continuous discharge on the EMG (electromyography) and degeneration of muscle. The discharge was blocked by treatment with curare. Thus, the rescued mice are useful to study the mechanism of myotonia and to identify the role of perlecan in adult tissue functions and diseases. Using the mutant mice, we found that in the absence of perlecan, tumor growth and angiogenesis were increased but tumor metastasis was inhibited in an experimental lung metastasis model in mice. We also found that skin wound healing was significantly accelerated in mutant mice. Our results suggest that perlecan plays important roles in cellular processes in various adult tissues and repair processes.

基底膜是薄的细胞外基质，其将上皮细胞和间充质细胞分开并包围细胞，例如内皮细胞、肌肉细胞和神经细胞。基底膜是发育过程中出现的第一种细胞外基质，对器官发育和组织修复至关重要。它们不仅为细胞和细胞层提供支架，而且在影响细胞粘附、迁移、增殖和分化的组织形态发生中具有重要作用。基底膜在血管中为蛋白质的通过和转移性肿瘤细胞的侵袭提供主要屏障。基底膜由IV型胶原、层粘连蛋白、串珠蛋白聚糖、巢蛋白/巢蛋白和其他分子组成，它们彼此相互作用以形成超分子结构，并且还结合细胞表面受体，如整联蛋白和多配体蛋白聚糖。这些蛋白质相互作用的破坏和基底膜蛋白表达的失调导致受损的组织发育和疾病。 层粘连蛋白是一个大的多结构域糖蛋白家族，通常特异于基底膜。迄今为止，已经鉴定了至少15种层粘连蛋白同种型，层粘连蛋白-1至层粘连蛋白-15。层粘连蛋白具有多种生物学活性，包括促进细胞粘附、迁移、分化、肿瘤细胞侵袭以及与基质分子和细胞表面受体的相互作用。层粘连蛋白α 1的LG 4模块是层粘连蛋白-1的一个亚基，在早期胚胎发育中起着关键作用。我们先前鉴定了来自层粘连蛋白α 1 LG 4的活性肽AG 73和EF-1，分别与肝素/多配体聚糖和整合素α 2 β 1结合。 然而，它们在层粘连蛋白-1蛋白和LG 4模块内的活性和功能关系尚不清楚。为了解决这个问题，我们制备了重组LG 4蛋白，这些蛋白在AG 73、EF-1和α-肌营养不良蛋白聚糖结合位点内含有位点特异性突变，并分析了它们与多配体聚糖、整合素和肝素的结合活性以及它们对细胞附着和扩散的活性，并将它们与层粘连蛋白-1进行了比较。我们发现在AG 73或α-肌营养不良蛋白聚糖结合位点内含有突变的重组蛋白失去肝素结合活性，并且不附着于表达个体多配体蛋白聚糖的淋巴细胞系，这表明LG 4通过这些位点结合多配体蛋白聚糖。这些突变的LG 4蛋白质显著降低了成纤维细胞附着，而EF-1中的突变的LG 4保留了细胞附着活性，但不促进细胞铺展。 成纤维细胞附着LG 4抑制肝素，但不整联蛋白抗体，而细胞扩散抑制抗整联蛋白α 2，但不α 6。 相反，层粘连蛋白-1介导的成纤维细胞附着和铺展不受肝素或抗整合素α 2的抑制，表明LG 4和层粘连蛋白-1具有不同的细胞附着和铺展机制。 这些结果表明，与其他层粘连蛋白α链类似，层粘连蛋白α 1 LG 4 -5也可以通过层粘连蛋白α 1链在其发挥其活性的某些组织中的蛋白水解裂解而产生。 对基因敲除小鼠和人类遗传疾病的研究表明，串珠素对发育至关重要，缺乏串珠素会导致由于心肌基底膜缺陷导致的胚胎死亡或由于软骨缺陷导致的围产期死亡。此外，串珠素涉及组织稳态和疾病中的许多生物学功能。我们之前创建了串珠素基因敲除（Perl-/-）小鼠，这些小鼠出现了严重的软骨发育不良，并在胚胎或围产期死亡。我们随后确定了两种人类疾病中串珠蛋白基因的突变，即以致死性软骨发育不良为特征的发育不良型（DDSH）和以肌强直和轻度软骨发育不良为特征的Schwartz-Jampel综合征（SJS）。动物模型可用于确定串珠素在成人组织功能和疾病中的作用。然而，Perl-/-小鼠的致死表型阻碍了这些研究。 为了克服这个问题，我们通过表达特异性针对软骨而非其他组织的重组串珠素来创建围产期致死性挽救的Perl-/-小鼠。突变小鼠存活并发展为肌强直，在EMG（肌电图）上显示持续放电和肌肉变性。用箭毒治疗后，排出物被阻断。因此，获救的小鼠可用于研究肌强直的机制，并确定串珠素在成人组织功能和疾病中的作用。使用突变小鼠，我们发现在不存在串珠素的情况下，在小鼠的实验性肺转移模型中，肿瘤生长和血管生成增加，但肿瘤转移被抑制。我们还发现，皮肤伤口愈合显着加快突变小鼠。我们的研究结果表明串珠素在各种成体组织的细胞过程和修复过程中起着重要作用。