Basement Membranes and Associated Protein Factors In Development and Disease

Dental & Craniofacial Research

Investigators

Linked publications & trials

Abstract

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. [unreadable] [unreadable] 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.[unreadable] [unreadable] 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.