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. Laminins are a family of large multidomain glycoproteins that are usually specific to basement membranes. Laminins perform a variety of biological activities, including promotion of cell adhesion, migration, differentiation, tumor cell invasion, and interactions with matrix molecules and cell surface receptors. Laminin-1 (also called laminin-111) is a heterotrimeric extracellular matrix protein composed of α1, β1, and γ1 chains and is crucial for early basement membrane assembly and embryonic implantation and development. Laminin-1 promotes neurite outgrowth in various neuronal cells, and several active sites in the α1 and γ1 chains for neurite outgrowth have been identified. In the nervous system, laminin-1 is expressed in the developing brain in a time- and region-specific manner. Laminin-1 is also expressed in the peripheral nervous system during development and in response to injury. Neurite outgrowth is a key event in differentiation of neuronal cells and is regulated by extracellular environmental factors, including cell adhesion molecules, such as laminin, and neurotrophic factors, such as nerve growth factor (NGF). The binding of laminin-1 activates integrins via a conformational change, which promotes neurite outgrowth. NGF signaling causes neurite outgrowth in neuronal cells but the effect is not sufficient, and laminin-dependent activation of integrins enhances its potential. Complete deficiency of laminin α1 (Lama1) in mice causes early embryonic lethality around embryonic day 7 (E7). Mutant mice expressing a truncated Lama1 lacking the C-terminal LG4 and LG5 subdomains die before E6.5. A missense mutation and conditional knockout of the Lama1 gene in mice disrupt retinal vascular development and inner limiting membrane formation. Although Lama1 is present in the meninges and in larger vessels in the late developmental stages of the central nervous system (CNS), the in vivo role of Lama1 in the CNS is unknown. In collaboration with Dr. Eri Arikawa-Hirasawa, we created conditional Lama1 knockout (Lama1-CKO) mice using epiblast-specific Sox2-Cre. Lama1-CKO mice survived postnatally. RT-PCR analysis revealed the absence of Lama1 mRNA in the cerebellum of mutant mice. Lama1-CKO mice survived but displayed impaired formation of the cerebellum and behavioral abnormalities when measured by the tail suspension test, the rotarod test, and footprint analysis. In the tail suspension test, Lama1-CKO mice showed hugging behavior. In the rotarod test, the movement time of Lama1-CKO mice was slower than that of control mice, indicating that Lama1-CKO mice have motor deficits. In the footprint test, Lama1-CKO mice showed a significant reduction in stride length and an increase in interlimb coordination when compared with control mice, indicating a locomotion disorder in Lama1-CKO mice. These behavioral abnormalities suggest that there are defects in neuronal functions, which result in the disequilibrium and lack of coordination seen in Lama1-CKO mice. In Lama1-CKO mice, LamA1 was absent in the pial basement membrane of the meninges, which resulted in defects in the conformation of the meninges. During cerebellar development, Lama1 deficiency caused abnormal migration and a decrease in the number of granule cell precursors, as well as disorganization of Bergmann glial fibers and endfeet, and a transient reduction in Akt activity. A marked reduction of dendritic processes in Purkinje cells was also observed in Lama1-CKO mice. These results revealed that Lama1 is critical for cerebellar development and function. Perlecan is a major heparan sulfate proteoglycan found in basement membranes and in other tissues such as cartilage. In skeletal muscle, perlecan is present in basement membranes surrounding muscle fibers and is particularly enriched in the neuromuscular region. Deficiency of perlecan causes lethal chondrodysplasia in both mice and humans. Perlecan mutations in patients with Schwartz-Jampel syndrome (SJS) result in myotonia and mild chondrodysplasia. Previously, we found that perlecan is essential for neuromuscular junction function. These results indicated that perlecan is important in muscle function. However, the role of perlecan in homeostasis of adult skeletal muscle is unclear. Perl-/- mice die perinatally due to defects in the tracheal cartilage. To overcome this problem, we created perinatal lethality-rescued Perl-/- (Perl-/-;Tg+/-) mice by expressing the perlecan transgene specifically in cartilage, under the control of the Col2a1 promoter. The mutant Perl-/-;Tg+/- mice survived and developed myotonia. Thus, the rescued mice are useful in studying the role of perlecan in adult tissues and diseases. Skeletal muscle is a dynamic tissue with a remarkable ability to maintain and regenerate in response to environmental stimuli that induce loading or injury. During these responses, for example after exercise, expression of local growth factors such as myostatin are enhanced and affect muscle growth. In collaboration with Dr. Eri Arikawa-Hirasawa, a former postdoc, we used a mouse model (Perl-/-;Tg+/-) deficient in skeletal muscle perlecan to study the role of perlecan in skeletal muscle hypertrophy, as well as in myostatin signaling with and without mechanical stress. Skeletal muscle can be classified into two broad fiber types: slow twitch Type I fibers and fast twitch Type II fibers. Type I muscle can sustain aerobic activity over prolonged periods. Type II fibers are more efficient for short bursts of speed and power. We found that the cross-sectional area of Type IIb fibers in tibialis anterior muscles was significantly increased in mutant mice compared to control mice. Mutant mice also had an increased number of type IIx fibers. In mutant mice, myostatin expression and its signaling were decreased. To examine the effects of mechanical overload or unload on fast and slow muscles in mutant mice, we performed tenotomy of the plantaris (fast) muscle and soleus (slow) muscle. Mechanical overload of the plantaris muscle significantly increased muscle wet weights in the mutant mice compared to control mice, and unloaded plantaris muscles caused less decrease in wet weights of mutant mice compared to WT mice. The decrease in myostatin expression in the overloaded plantaris muscle was profound and significant in mutant mice compared to WT mice. In contrast, overloading soleus muscles caused no changes in either type of muscle. These results suggest that perlecan is critical for maintaining fast muscle mass and fiber composition and for regulating myostatin signaling.

Project Start
Project End
Budget Start
Budget End
Support Year
23
Fiscal Year
2011
Total Cost
$690,863
Indirect Cost
Name
National Institute of Dental & Craniofacial Research
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