Basement membranes (BMs) are thin extracellular matrices (ECMs) that separate epithelial and mesenchymal cells and surround other cells, such as endothelial, muscular, and neural cells. BMs consist of a unique set of proteins, such as laminins, perlecan, and collagen IV. They are also associated with other ECM proteins, such as fibulin and fibronectin, in various tissues. BMs are the first ECMs to appear during development and are critical for organ development and tissue repair. They provide the scaffold for cells and cell layers and play essential roles in cell adhesion, migration, proliferation, and differentiation during morphogenesis. We have studied the specific functions of basement membranes and their associated proteins to delineate their structural and functional relationships, to elucidate their regulatory mechanisms, and to describe related protein interactions that occur during development and disease. A C-terminal fibulin-7 fragment (Fbln7-C) shows anti-angiogenesis activity in vitro: Fibulins comprise a family of seven secreted glycoproteins that are associated with basement membranes, elastic fibers, and other matrices. Fibulins mediate cell-to-cell and cell-to-matrix communication, as well as stabilize the ECM during organogenesis. Fibulins have been implicated in the modulation of cell morphology, growth, adhesion, and motility. Fibulins also act as both tumor-suppressors and oncogenic factors. We previously identified fibulin-7 (Fbln7)/TM14, the newest member of the fibulin family, as being expressed by odontoblasts as a cell adhesion molecule for dental mesenchyme cells and odontoblasts. We also found that Fbln7 interacts with ECM proteins and growth factors. It has been shown that fragments of several ECM proteins, such as endostatin, angiostatin, and endorepellin, have negative effects on angiogenesis. Using human umbilical vein endothelial cells (HUVECs), we found that recombinant Fbln7 inhibited endothelial cell tube formation. We showed that a Fbln7-C-terminal fragment (Fbln7-C) had the strongest inhibitory effect on HUVEC tube formation, and on aortic ring vessel sprouting in vitro. Fbln7-C binds to HUVECs through alpha5beta1 integrin, and it inhibits actin stress fiber formation. Fbln7-C induces alpha5beta1 integrin clustering at cell adhesion sites with other focal adhesion molecules and sustained activation of FAK, p130Cas, and Rac1. In addition, RhoA activation was decreased, thereby inhibiting HUVEC spreading and migration. Our findings suggest that Fbln7-C is a novel anti-angiogenic factor. Perlecan inhibits autophagy to maintain muscle homeostasis in mouse soleus muscle: Perlecan is a major heparan sulfate proteoglycan in basement membranes. Skeletal muscle continuously adapts in response to a variety of stimuli. Mechanical load causes hypertrophy, while physical inactivity leads to atrophy. Muscle mass is in a dynamic equilibrium between protein synthesis and protein degradation. The autophagy-lysosome system is essential for muscle protein synthesis and degradation equilibrium, and its dysfunction has been linked to various muscle disorders. In collaboration with Dr. Eri Arikawa-Hirasawa, we studied the association between perlecan and autophagy in the slow-twitch soleus muscle using a lethality-rescued perlecan knockout mouse model (Hspg2-/-; Tg), in which the perlecan transgene is expressed specifically in cartilage but not other tissues, including muscles. We found that perlecan deficiency in slow-twitch soleus muscles enhanced autophagic activity. This enhancement was accompanied by a decrease in autophagic substrates, and an increase in LC3II levels. Perlecan deficiency also caused a reduction in the phosphorylation levels of p70S6k and Akt and increased the phosphorylation of AMPK. Our findings revealed that perlecan inhibits the autophagic process through activation of the mTORC1 pathway. This autophagic response may be a novel target for enhancing the efficacy of skeletal muscle atrophy treatment.

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Project End
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Budget End
Support Year
27
Fiscal Year
2015
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Dental & Craniofacial Research
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Sadatsuki, Ryo; Kaneko, Haruka; Kinoshita, Mayuko et al. (2017) Perlecan is required for the chondrogenic differentiation of synovial mesenchymal cells through regulation of Sox9 gene expression. J Orthop Res 35:837-846
de Vega, Susana; Hozumi, Kentaro; Suzuki, Nobuharu et al. (2016) Identification of peptides derived from the C-terminal domain of fibulin-7 active for endothelial cell adhesion and tube formation disruption. Biopolymers 106:184-195
Ning, Liang; Xu, Zhuo; Furuya, Norihiko et al. (2015) Perlecan inhibits autophagy to maintain muscle homeostasis in mouse soleus muscle. Matrix Biol 48:26-35
Ning, Liang; Kurihara, Hidetake; de Vega, Susana et al. (2014) Laminin ?1 regulates age-related mesangial cell proliferation and mesangial matrix accumulation through the TGF-? pathway. Am J Pathol 184:1683-94
Kerever, Aurelien; Mercier, Frederic; Nonaka, Risa et al. (2014) Perlecan is required for FGF-2 signaling in the neural stem cell niche. Stem Cell Res 12:492-505
Suzuki, Nobuharu; Numakawa, Tadahiro; Chou, Joshua et al. (2014) Teneurin-4 promotes cellular protrusion formation and neurite outgrowth through focal adhesion kinase signaling. FASEB J 28:1386-97
de Vega, Susana; Suzuki, Nobuharu; Nonaka, Risa et al. (2014) A C-terminal fragment of fibulin-7 interacts with endothelial cells and inhibits their tube formation in culture. Arch Biochem Biophys 545:148-53
Yoshizaki, Keigo; Yamada, Yoshihiko (2013) Gene evolution and functions of extracellular matrix proteins in teeth. Orthod Waves 72:1-10
Kaneko, Haruka; Ishijima, Muneaki; Futami, Ippei et al. (2013) Synovial perlecan is required for osteophyte formation in knee osteoarthritis. Matrix Biol 32:178-87
Arakaki, Makiko; Ishikawa, Masaki; Nakamura, Takashi et al. (2012) Role of epithelial-stem cell interactions during dental cell differentiation. J Biol Chem 287:10590-601

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