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。基底膜是发育过程中最先出现的细胞外基质，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，而且在影响细胞粘附、迁移、增殖和分化的形态发生中起着重要作用。基底膜由IV型胶原蛋白、层粘连蛋白、perlecan、nidogen/entactin等分子组成，这些分子相互作用形成超分子结构。

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.