Yuta Fukai1, Yutaka Ohsawa1, Hideaki Ohtsubo1, Shin-Ichiro Nishimatsu2, Hiroki Hagiwara3, Makoto Noda4, Toshikuni Sasaoka5, Tatsufumi Murakami1, Yoshihide Sunada6. 1. Department of Neurology, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan. 2. Department of Natural Science, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan. 3. Department of Medical Science, Teikyo University of Science, 2-2-1 Senjusakuragi, Adachi-ku, Tokyo 120-0045, Japan. 4. Department of Molecular Oncology, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. 5. National Institute for Basic Biology, Okazaki 444-8585, Japan; Department of Laboratory Animal Science, Kitasato University School of Medicine, Sagamihara 252-0374, Japan; Department of Comparative and Experimental Medicine, Center for Bioresource-based Researches, Brain Research Institute, Niigata University, Niigata 951-8585, Japan. 6. Department of Neurology, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan. Electronic address: ysunada@med.kawasaki-m.ac.jp.
Abstract
BACKGROUND: The dystroglycan complex consists of two subunits: extracellular α-dystroglycan and membrane-spanning β-dystroglycan, which provide a tight link between the extracellular matrix and the intracellular cytoskeleton. Previous studies showed that 43 kDa β-dystroglycan is proteolytically cleaved into the 30 kDa fragment by matrix metalloproteinases (MMPs) in various non-muscle tissues, whereas it is protected from cleavage in muscles by the sarcoglycan complex which resides close to the dystroglycan complex. It is noteworthy that cleaved β-dystroglycan is detected in muscles from patients with sarcoglycanopathy, sarcoglycan-deficient muscular dystrophy. In vitro assays using protease inhibitors suggest that both MMP-2 and MMP-9 contribute to the cleavage of β-dystroglycan. However, this has remained uninvestigated in vivo. METHODS: We generated triple-knockout (TKO) mice targeting MMP-2, MMP-9 and γ-sarcoglycan to examine the status of β-dystroglycan cleavage in the absence of the candidate matrix metalloproteinases in sarcoglycan-deficient muscles. RESULTS: Unexpectedly, β-dystroglycan was cleaved in muscles from TKO mice. Muscle pathology was not ameliorated but worsened in TKO mice compared with γ-sarcoglycan single-knockout mice. The gene expression of MMP-14 was up-regulated in TKO mice as well as in γ-sarcoglycan knockout mice. In vitro assay showed MMP-14 is capable to cleave β-dystroglycan. CONCLUSIONS: Double-targeting of MMP-2 and MMP-9 cannot prevent cleavage of β-dystroglycan in sarcoglycanopathy. Thus, matrix metalloproteinases contributing to β-dystroglycan cleavage are redundant, and MMP-14 could participate in the pathogenesis of sarcoglycanopathy.
BACKGROUND: The dystroglycan complex consists of two subunits: extracellular α-dystroglycan and membrane-spanning β-dystroglycan, which provide a tight link between the extracellular matrix and the intracellular cytoskeleton. Previous studies showed that 43 kDa β-dystroglycan is proteolytically cleaved into the 30 kDa fragment by matrix metalloproteinases (MMPs) in various non-muscle tissues, whereas it is protected from cleavage in muscles by the sarcoglycan complex which resides close to the dystroglycan complex. It is noteworthy that cleaved β-dystroglycan is detected in muscles from patients with sarcoglycanopathy, sarcoglycan-deficient muscular dystrophy. In vitro assays using protease inhibitors suggest that both MMP-2 and MMP-9 contribute to the cleavage of β-dystroglycan. However, this has remained uninvestigated in vivo. METHODS: We generated triple-knockout (TKO) mice targeting MMP-2, MMP-9 and γ-sarcoglycan to examine the status of β-dystroglycan cleavage in the absence of the candidate matrix metalloproteinases in sarcoglycan-deficient muscles. RESULTS: Unexpectedly, β-dystroglycan was cleaved in muscles from TKO mice. Muscle pathology was not ameliorated but worsened in TKO mice compared with γ-sarcoglycan single-knockout mice. The gene expression of MMP-14 was up-regulated in TKO mice as well as in γ-sarcoglycan knockout mice. In vitro assay showed MMP-14 is capable to cleave β-dystroglycan. CONCLUSIONS: Double-targeting of MMP-2 and MMP-9 cannot prevent cleavage of β-dystroglycan in sarcoglycanopathy. Thus, matrix metalloproteinases contributing to β-dystroglycan cleavage are redundant, and MMP-14 could participate in the pathogenesis of sarcoglycanopathy.