Basement membranes are thin extracellular matrices that separate epithelial and mesenchymal cells, and surround cells such as endothelial, muscle, 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 play 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, and they interact with one another to form the supramolecular structure. Perlecan is a major heparan sulfate proteoglycan in basement membranes, and in some other tissues such as cartilage. Perlecan interacts with many extracellular molecules, growth factors and cell surface receptors, and is implicated in many biological functions of 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 10.5, and those that survive die perinatally because of a respiratory problem due to defective tracheal cartilage. 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), which is characterized by lethal chondrodysplasia, and Schwartz-Jampel syndrome (SJS), which is characterized by myotonia and milder chondrodysplasia. When we examined newborn Perl-/- mice, we noticed that cross sections of the skeletal muscle in Perl-/- mice were larger than those of control mice. Since perlecan surrounds skeletal muscles, we hypothesized that perlecan may play a role in muscle homeostasis in adult mice. However, the lethal phenotype of Perl-/- mice has hampered these studies, and so to overcome this problem, we created perinatal lethality-rescued Perl-/- (Perl-/-;TgPerl) mice by expressing the perlecan transgene specifically in cartilage under the control of the Col2a1 promoter. The mutant mice survived and developed myotonia, lacking perlecan in the skeletal muscle. In collaboration with Dr. Eri Arikawa-Hirasawa, a former member of the lab, we examined adult skeletal muscle hypertrophy, as well as myostatin expression and its signaling pathway with and without mechanical stress created by tenotomy of cooperative muscle in the hindlimbs of this mouse model. We found that the fiber cross-sectional area (CSA) of myosin heavy chains (MHC) type IIb and type IIx fibers in the tibialis anterior (TA, fast) muscles was significantly increased in the mutant mice. The number of fibers in the TA muscle was also increased. Myostatin, a negative regulator of muscle growth, and its type I receptor (ALK4) expression were substantially decreased. Myostatin-induced Smad activation was also reduced in a culture of myotubes from the muscle of the mutant mice, suggesting that myostatin expression and its signaling were decreased in the perlecan-deficient muscle. To examine the effects of mechanical overload or unload on fast and slow muscles in the mutant mice, we performed tenotomy of the plantaris (fast) muscle and soleus (slow) muscle. Mechanical overload on the plantaris muscle of the mutant mice significantly increased wet weights and the CSA, and changed the fiber composition in comparison to that of wild type (WT) mice. Unloaded plantaris muscles of the mutant mice caused less decrease in wet weights compared to those of WT mice. In contrast, overloading soleus muscles caused no changes in either type of muscle. Our results suggest that perlecan plays an important role in maintaining skeletal muscle, and is a regulator for maintaining muscle fiber types in response to uploading stress. Laminin consists of 3 chains, α, β, and γ. To date, 15 laminin isoforms have been identified, and 12 genetically distinct laminin chains (α1-5, β1-4, and γ1-3) are known to exist. The αchains contain a large C-terminal globular domain (G-domain) with 5 internal repeats (LG1-5). Laminin has a variety of biological activities, including promotion of cell adhesion, migration, differentiation, tumor cell invasion, and interaction with matrix molecules and cell surface receptors, including integrins, syndecans and α-dystroglycan. The laminin α2 chain (Lama2), a component of several laminin isoforms (Lam-211, -221, and -213), is expressed in skeletal muscle, peripheral nerves, brain, capillaries and submandibular glands. The G-domain of Lama2 is implicated in providing a linkage between the extracellular matrix and cells, through its interaction with cell surface receptors such as heparan sulfate proteoglycans and α-dystroglycan. In collaboration with Dr. Motoyoshi Nomizu, we identified heparin and α-dystroglycan binding sequences in the LG45 module of Lama2 using synthetic peptides and recombinant proteins. Addition of the binding peptides inhibited submandibular gland development in explant culture. Our results suggest that these active sites are critical for biological activity of Lama2.
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