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.
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