Gene Regulation And Function Of Basement Membranes

基底膜的基因调控和功能

• 批准号: 7146109
• 负责人: Yoshihiko Yamada
• 金额: --
• 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/7146109
• 关键词: basement membrane; dental development; disease /disorder model; gene targeting; genetic regulation; genetically modified animals; heparan sulfate; histogenesis; human tissue; laboratory mouse; laminin; membrane activity; membrane structure; model design /development; myotonic dystrophy; protein protein interaction; proteoglycan

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 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. Recently, genetic diversity in the subunits of laminin and type IV collagen has been found and the existence of a large family of these molecules has been demonstrated. Our primary objectives have been to identify the specific functions of basement membrane components, to study their structure and function relationships, to elucidate the mechanisms by which they are regulated, and to describe related protein interactions in development and diseases. We have created animal models to study functions of basement membrane components in development and disease. 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 promoting cell adhesion, migration, differentiation, tumor cell invasion, and interactions with matrix molecules and cell surface receptors. We previously identified active sites of the laminin alpha-1 chain, a subunit of laminin-1, using synthetic peptides. To identify the in vivo function of the laminin alpha-1 chain (Lama1), we created conventional knockout and conditional knockout mice. Complete deficiency of Lama1 caused early embryonic lethality around embryonic day 8.5 (E8.5). In mutant embryos, the embryonic basement membrane was formed, but Reichert?s membrane was completely absent and formation of the extraembryonic ectoderm was severely defective. After E6.5, the cell polarity of visceral endoderm was lost, and the cells formed a disorganized, multilayered structure and failed to form mature anterior visceral endoderm. Epiblasts formed as polarized epithelia at E5.5, but at later stages, proximal epiblasts did not migrate to form the primitive streak. Mice with a conditional deficiency of Lama1 in epiblasts survived. These results suggest that Lama1 expressed by parietal and visceral endoderm is a functional and structural component of the embryonic basement membrane and Reichert?s membrane and plays a critical role in the development of the extraembryonic structure that is essential for differentiation and migration of embryonic ectoderm and mesoderm. Currently, we have been studying the role of Lama1 in salivary gland development and other tissue functions in adult using conditional knockout mice. Laminin alpha5 (Lama5), a component of laminin-10/11, is the major laminin alpha chain in tooth basement membrane. We examined the role of Lama5 in early tooth morphogenesis. Lama5-null mice develop a small tooth germ with no cusps, in which the inner dental epithelium is not polarized and enamel knot formation is defective. Integrin alpha-6/beta-4, a ligand of Lama5 is not localized at the basal layer of the epithelium but diffusely expressed around the cell surface. We found that laminin 10/11 promotes spreading and filopodia-like micro-spike formation of dental epithelium. Inhibition studies using cell and tooth germ organ cultures suggest that the interaction of Lama5 and Integrin alpha-6/beta-4 mediates these cellular changes through PI 3 kinase-Cdc42/Rac signaling. These studies demonstrate that Lama5 regulates the polarity and formation of the monolayer of the inner dental epithelium and that these cellular processes are essential for tooth growth and morphogenesis. We previously created perlecan knockout mice, which developed a severe chondrodysplasia with micromelia and died as embryos or perinatally. We subsequently identified functional-null mutations of perlecan, which cause a lethal chondrodysplasia in humans, dyssegmental dysplasia, Silverman-Handmaker type (DDSH). We also identified partially functional mutations of perlecan in a milder human genetic disorder Schwartz-Jampel syndrome (SJS), characterized by myotonia and chondrodysplasia. In order to define the mechanism of myotonia caused by the defect in perlecan, we have rescued the perinatal lethality of perlecan-null mice by expressing recombinant perlecan specifically in cartilage. The 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 develop therapeutic agents for the disease. Perlecan is implicated in atherosclerosis, because lipoproteins associate with proteoglycans. In collaboration with Dr. Ira Goldberg, we showed that heterozygous perlecan mice developed less atherosclerosis compared to wild-type mice.

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、增殖和分化的形态发生中起重要作用。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、串珠素、巢蛋白/巢蛋白和其他分子组成，它们相互作用形成超分子结构。最近，已发现层粘连蛋白和IV型胶原蛋白亚基的遗传多样性，并且已证明存在这些分子的大家族。我们的主要目标是确定基底膜成分的特定功能，研究它们的结构和功能关系，阐明它们的调节机制，并描述发育和疾病中相关的蛋白质相互作用。我们已经建立了动物模型来研究基底膜成分在发育和疾病中的功能。 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，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。我们以前确定了层粘连蛋白α-1链的活性位点，层粘连蛋白-1的亚基，使用合成肽。为了鉴定层粘连蛋白α-1链（Lama 1）的体内功能，我们创建了常规敲除和条件性敲除小鼠。Lama 1的完全缺乏导致胚胎在胚胎8.5天（E8.5）左右早期死亡。在突变胚胎中，胚胎基底膜形成，但Reichert？s膜完全缺失，胚外外胚层的形成严重缺陷。E6.5以后，内脏内胚层的细胞极性丧失，细胞形成紊乱的多层结构，不能形成成熟的前内脏内胚层。上胚层在E5.5形成极化上皮，但在后期，近端上胚层没有迁移形成原始条纹。外胚层中Lama 1条件性缺乏的小鼠存活。这些结果表明，Lama 1表达的壁和内脏内胚层是一个功能和结构组成部分的胚胎基底膜和Reichert？在胚胎外胚层和中胚层的分化和迁移所必需的胚外结构的发育中起着关键作用。目前，我们一直在研究Lama 1在唾液腺发育和其他组织功能的成年条件敲除小鼠中的作用。 层粘连蛋白α 5（Lama 5）是层粘连蛋白-10/11的组分，是牙齿基底膜中的主要层粘连蛋白α链。我们研究了Lama 5在早期牙齿形态发生中的作用。Lama 5基因敲除小鼠发育出一个没有尖突的小牙胚，其中内牙上皮没有极化，釉质结形成有缺陷。整联蛋白α-6/β-4（Lama 5的配体）不定位于上皮的基底层，而是在细胞表面周围弥散表达。我们发现层粘连蛋白10/11促进牙上皮细胞的铺展和丝状伪足样微刺的形成。使用细胞和牙胚器官培养物的抑制研究表明，Lama 5和整联蛋白α-6/β-4的相互作用通过PI 3激酶-Cdc 42/Rac信号传导介导这些细胞变化。这些研究表明，Lama 5调节内牙上皮细胞单层的极性和形成，这些细胞过程对牙齿生长和形态发生至关重要。 我们以前创造了串珠素基因敲除小鼠，这些小鼠发展为严重的软骨发育不良伴小肢畸形，并在胚胎或围产期死亡。随后，我们确定了串珠素的功能无效突变，其导致人类致命的软骨发育不良，发育不良，Silverman-Handmaker型（DDSH）。我们还确定了部分功能性突变串珠蛋白在一个温和的人类遗传性疾病施瓦茨-詹佩尔综合征（SJS），其特征是肌强直和软骨发育不良。为了确定由串珠素缺陷引起的肌强直的机制，我们通过在软骨中特异性表达重组串珠素来挽救串珠素缺失小鼠的围产期致死率。小鼠存活并发展为肌强直，在EMG（肌电图）上显示连续放电和肌肉变性。用箭毒治疗后，排出物被阻断。因此，拯救的小鼠可用于研究肌强直的机制和开发用于该疾病的治疗剂。串珠素与动脉粥样硬化有关，因为脂蛋白与蛋白聚糖有关。在与伊拉戈德堡博士的合作中，我们发现杂合串珠蛋白聚糖小鼠比野生型小鼠发生动脉粥样硬化的程度更低。