Basement Membranes and Associated Protein Factors In Development and Disease

基底膜是薄的细胞外基质，分隔上皮细胞和间充质细胞，并包围细胞，如内皮细胞、肌肉细胞和神经细胞。基底膜是发育中第一个出现的细胞外基质，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，而且在影响细胞黏附、迁移、增殖和分化的形态发生中也起着至关重要的作用。基底膜由IV型胶原蛋白、层粘连蛋白、粘附素、粘附素/牙本质蛋白等分子组成，它们相互作用形成超分子结构。 Perlecan是一种主要的硫酸乙酰肝素蛋白多糖，存在于基底膜和其他一些组织中，如软骨。Perlecan由一个400 kDa的蛋白质核心组成，可分为5个不同的结构域，命名为I-V。蛋白质核心在N-末端和C-末端含有共价连接的糖胺聚糖(GAG)链。Perlecan与细胞外分子、生长因子和细胞表面受体相互作用，参与组织发育、动态平衡和疾病的许多生物学功能。为了确定perlecan在发育中的作用，我们之前创建了perlecan基因敲除(perl-/-)小鼠。一些Perl-/-小鼠在胚胎10.5天左右死亡，而那些幸存下来的小鼠由于呼吸问题而在围产期死亡。存活到围产期的Perl-/-小鼠表现出软骨发育缺陷的侏儒症。我们之前在两种人类疾病中发现了perlecan基因的突变：以致死性软骨发育不良为特征的Silverman-Handaker型(DDSH)，以及以肌强直和轻度软骨发育不良为特征的Schwartz-Jampel综合征(SJS)。Perlecan缺乏会导致小鼠和人类致死性软骨发育不良，这表明Perlecan对软骨发育是必不可少的。然而，Perlecan在软骨发育中的作用尚不清楚。Perl-/-生长板较短，软骨内骨形成严重受损。我们发现，在生长板形成过程中，Perlecan在成纤维细胞生长因子/血管内皮生长因子信号转导和血管侵袭中起着关键作用。在Perl-/-生长板中，FGFR1和FGFR3均被激活。增生性软骨细胞上调了成纤维细胞生长因子/成纤维细胞生长因子受体的靶基因--血管内皮生长因子的表达，提示肥厚区缺乏血管生成不是由于血管内皮生长因子表达降低所致。我们的结果表明，软骨内骨化受损的部分原因是在没有骨钙素的情况下，软骨内血管侵入软骨的缺陷。 动物模型有助于确定Perlecan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，我们创造了围产期致死性拯救的Perl-/-(Perl-/-；TgPerl)小鼠，在Col2a1启动子的控制下，将Perlecan转基因特异地表达到软骨中。突变小鼠存活并出现肌强直，肌电持续放电，肌肉变性，可用于开发治疗肌强直的药物。Perlecan与血管生成、伤口修复、肿瘤生长和转移有关。Perlecan在这些生物过程中的活性经常因研究所用的组织和细胞类型而异，有时会有争议，部分原因是这些实验系统中存在野生型或突变型Perlecan。因此，Perl-/-；TgPerl小鼠在大多数组织中缺乏perlecan，应该有助于确定perlecan在发病机制和组织再生中的真正作用。为了检测宿主Perlecan是否影响肿瘤生长，我们将Lewis肺癌肿瘤注射到Perl-/-；TgPerl小鼠皮下。我们发现Perl-/-；TgPerl小鼠的肿瘤生长速度比对照小鼠快。杂合子小鼠(Perl+/-；TgPerl)患上中等大小的肿瘤。这些数据表明，宿主Perlecan可以抑制肿瘤生长。我们发现，在皮肤穿孔试验中，突变小鼠的伤口愈合速度加快。肿瘤生长加快和伤口愈合加速伴随着血管生成的增加。这可能是由于在没有Perlecan的情况下生长因子的可用性和/或表达增加所致。同样可以想象的是，细胞可以在缺乏Perlecan的基质中更快地迁移。 在我们的Perlecan项目中，我们创造了表达重组Perlecan的转基因小鼠。我们发现，其中一只转基因小鼠在出生后4周左右出现了严重的后肢震颤。特发性震颤是一种人类神经疾病，当一个人移动或试图移动时，会在没有可识别原因的情况下发生震颤(颤抖)，是最常见的震颤障碍，也是世界范围内最普遍的运动障碍。它的发病率是帕金森氏症的20倍。诊断以临床表现为主，治疗以症状为主。由于震颤小鼠可能是了解特发性震颤障碍的有用动物模型，我们想要确定致病基因。我们发现转基因被插入到ODZ4基因的内含子14中。ODZ4是一种在中枢神经系统(CNS)中高表达的跨膜蛋白，但其功能尚不清楚。ODZ4和转基因在突变小鼠的中枢神经系统中不表达。缺乏ODZ4导致脊柱髓鞘过少，导致少突胶质细胞数量减少和神经元退化，这可能导致在纯合子突变小鼠中观察到的震颤表型。

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 consist of collagen IV, laminin, perlecan, nidogen/entactin, and other molecules, which interact with each other to form the supramolecular structure. Perlecan is a major heparan sulfate proteoglycan in basement membranes and in some other tissues, such as cartilage. Perlecan consists of a 400-kDa protein core, which can be divided into 5 distinct domains, designated I-V. The protein core contains covalently attached glycosaminoglycan (GAG) chains at the N- and C-terminal domains. Perlecan interacts with extracellular molecules, growth factors, and cell surface receptors and is implicated in many biological functions in tissue development, homeostasis, and disease. In order to identify the role of perlecan in development, we previously created perlecan knockout (Perl-/-) mice. Some Perl-/- mice die around embryonic day (E) 10.5, and those that survive die perinatally because of a respiratory problem. The Perl-/- mice that survive to the perinatal stage show dwarfism with defective cartilage development. We previously identified mutations of the perlecan gene in 2 human diseases: dyssegmental dysplasia, Silverman-Handmaker type (DDSH), characterized by lethal chondrodysplasia, and Schwartz-Jampel syndrome (SJS), characterized by myotonia and milder chondrodysplasia. Perlecan-deficiency causes lethal chondrodysplasia in mice and humans, indicating that perlecan is essential for cartilage development. However, the role of perlecan in cartilage development was unknown. The Perl-/- growth plate was short, with severely impaired endochondral bone formation. We found a critical role for perlecan in FGF/VEGF signaling and vascular invasion during growth plate formation. Both FGFR1 and FGFR3 were activated in the Perl-/- growth plate. Expression of VEGF, an FGF/FGFR target gene, was upregulated by Perl-/- hypertrophic chondrocytes, suggesting that the lack of vascularization into the hypertrophic zone is not due to reduced VEGF expression. Our results suggest that impaired endochondral ossification is in part due to defects of vascular invasion into cartilage in the absence of perlecan. 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 this problem, we created perinatal lethality-rescued Perl-/- (Perl-/-;TgPerl) mice by expressing the perlecan transgene specifically to cartilage under the control of the Col2a1 promoter. The mutant mice survived and developed myotonia, showing a continuous discharge by electromyography and degeneration of muscle and can be used to develop therapeutic reagents for myotonia. Perlecan is implicated in angiogenesis, wound repair, tumor growth, and metastasis. Activities of perlecan in these biological processes often vary depending on tissues and cell types used for the study and are sometimes controversial, in part because wild-type or mutant perlecan is present in these experimental systems. Therefore, Perl-/-;TgPerl mice, which lack perlecan in most tissues, should be useful for defining the real role of perlecan in pathogenesis and tissue regeneration. To examine whether the host perlecan affects tumor growth, we injected Lewis lung carcinoma tumors subcutaneously into Perl-/-;TgPerl mice. We found that tumor growth was faster in Perl-/-;TgPerl mice than in control mice. Heterozygous mice (Perl+/-;TgPerl) developed intermediate-sized tumors. These data suggest that host perlecan inhibits tumor growth. We found that wound healing was accelerated in mutant mice in skin-punch assays. Increased tumor growth and accelerated wound healing were accompanied by an increase in angiogenesis. This may be caused by an increase in availability and/or expression of growth factors in the absence of perlecan. It is also conceivable that cells can migrate faster in perlecan-deficient matrix. During our project on perlecan, we created transgenic mice expressing recombinant perlecan. We found that one of the transgenic mouse lines developed severe tremors in the hindlimbs around 4 weeks after birth. Essential tremor, a human nerve disorder in which tremors (shakes) occur without an identifiable cause when a person is moving or trying to move, is the most common tremor disorder, and it is the most prevalent movement disorder worldwide. It is 20 times more common than Parkinson's disease. The diagnosis is based on clinical findings, and treatment is mainly for symptoms. Since the tremor mice might be a useful animal model for understanding essential tremor disorder, we wanted to identify the causative gene. We found that the transgene was inserted into intron 14 of the Odz4 gene. Odz4 is a transmembrane protein that is highly expressed in the central nervous system (CNS), but its function is not known. Odz4 and the transgene were not expressed in the CNS of mutant mice. The absence of Odz4 resulted in hypomyelination in the spine, causing a reduced number of oligodendrocytes and degeneration of neurons, which likely led to the tremor phenotype observed in the homozygous mutant mice.