Calpain-6 deficiency promotes skeletal muscle development and regeneration.
Calpain-6 deficiency promotes skeletal muscle development and regeneration.
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DOI：
10.1371/journal.pgen.1003668
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发表时间：
2013
期刊：
影响因子：
4.5
通讯作者：
Sorimachi H
中科院分区：
文献类型：
--
作者：
Tonami K;Hata S;Ojima K;Ono Y;Kurihara Y;Amano T;Sato T;Kawamura Y;Kurihara H;Sorimachi H
钙蛋白酶是一种钙离子依赖性的半胱氨酸蛋白酶，在几乎所有的真核生物中都具有多种功能。有超过10种高度保守的哺乳动物钙蛋白酶，其中真兽钙蛋白酶-6（CAPN 6）是独特的，因为它在活性位点Cys残基（人类中为Lys）处具有氨基酸取代，强烈暗示蛋白水解活性的丧失。CAPN 6主要在胚胎肌肉、胎盘和几种培养的细胞系中表达。我们以前报道过CAPN 6参与调控培养细胞中微管动力学和肌动蛋白重组。然而，CAPN 6的生理功能仍然不清楚。在这里，为了阐明CAPN 6的体内作用，我们产生了Capn 6缺陷小鼠，其中lacZ表达盒被整合到Capn 6基因中。这些Capn 6缺陷的小鼠胚胎主要在骨骼肌以及软骨和心脏中表达lacZ。组织学和生化分析表明，CAPN 6缺陷促进胚胎骨骼肌的发育。在诱导分化成肌管的原代培养的骨骼肌细胞中，在骨骼肌细胞中检测到Capn 6表达，并且Capn 6缺陷培养物显示出增加的分化。此外，我们发现CAPN 6在心脏毒素诱导的变性后的成年小鼠再生骨骼肌中表达。在该实验系统中，Capn 6缺陷型小鼠在同一时间点比杂合子或野生型小鼠表现出更先进的肌肉再生。这些结果共同表明，CAPN 6的缺失在发育和再生过程中促进骨骼肌分化，表明CAPN 6作为骨骼肌分化的抑制剂的新生理功能。钙蛋白酶是一个由钙离子调节的细胞内蛋白酶家族，具有不同的结构和功能。在哺乳动物钙蛋白酶中，钙蛋白酶-6是独特的“非蛋白水解”，其生理作用仍然未知。在这项研究中，我们使用Capn 6基因敲除小鼠，发现Calpain-6参与体内骨骼肌发育和再生。Calpain-6主要在胚胎肌肉和骨软骨中表达。Capn 6基因敲除促进了骨骼肌的发育，这是祖细胞分化增加的结果。此外，在再生骨骼肌中检测到钙蛋白酶-6，并抑制这一过程。这些结果表明哺乳动物钙蛋白酶-6是骨骼肌分化和生长的抑制性调节剂。我们的研究结果表明，钙蛋白酶-6可能作为一个治疗目标的肌营养不良/萎缩或作为一个有用的工具，在组织工程。对于钙蛋白酶研究，钙蛋白酶-6的生理非蛋白水解作用可能揭示了难以捉摸的钙蛋白酶功能，独立于他们的充分研究的蛋白水解活性。此外，由于这种结构上的非蛋白水解功能是唯一的脊椎动物中的胎盘哺乳动物的钙蛋白酶-6，我们的突变小鼠可能提供深入了解分子和生物进化之间的关系。
Calpains are Ca2+-dependent modulator Cys proteases that have a variety of functions in almost all eukaryotes. There are more than 10 well-conserved mammalian calpains, among which eutherian calpain-6 (CAPN6) is unique in that it has amino acid substitutions at the active-site Cys residue (to Lys in humans), strongly suggesting a loss of proteolytic activity. CAPN6 is expressed predominantly in embryonic muscles, placenta, and several cultured cell lines. We previously reported that CAPN6 is involved in regulating microtubule dynamics and actin reorganization in cultured cells. The physiological functions of CAPN6, however, are still unclear. Here, to elucidate CAPN6's in vivo roles, we generated Capn6-deficient mice, in which a lacZ expression cassette was integrated into the Capn6 gene. These Capn6-deficient mouse embryos expressed lacZ predominantly in skeletal muscles, as well as in cartilage and the heart. Histological and biochemical analyses showed that the CAPN6 deficiency promoted the development of embryonic skeletal muscle. In primary cultured skeletal muscle cells that were induced to differentiate into myotubes, Capn6 expression was detected in skeletal myocytes, and Capn6-deficient cultures showed increased differentiation. Furthermore, we found that CAPN6 was expressed in the regenerating skeletal muscles of adult mice after cardiotoxin-induced degeneration. In this experimental system, Capn6-deficient mice exhibited more advanced skeletal-muscle regeneration than heterozygotes or wild-type mice at the same time point. These results collectively showed that a loss of CAPN6 promotes skeletal muscle differentiation during both development and regeneration, suggesting a novel physiological function of CAPN6 as a suppressor of skeletal muscle differentiation. The calpains arose evolutionarily as a family of Ca2+-regulated intracellular proteases with divergent structures and functions. Unique among mammalian calpains, calpain-6 is “non-proteolytic”, and its physiological role has remained unknown. In this study, using Capn6 knock-out mice, we discovered that Calpain-6 is involved in skeletal muscle development and regeneration in vivo. Calpain-6 was predominantly expressed in embryonic muscles and bone cartilage. Capn6 knock-out promoted skeletal-muscle development as a result of increased progenitor cell differentiation. Furthermore, calpain-6 was detected in regenerating skeletal muscles, and suppressed this process. These results indicate that mammalian calpain-6 is a suppressive modulator for skeletal muscle differentiation and growth. Our findings indicate that calpain-6 might serve as a therapeutic target for muscular dystrophy/atrophy or as a useful tool in tissue engineering. For calpain studies, the physiological non-proteolytic role of calpain-6 may shed light on the elusive calpain functions that are independent of their well-studied proteolytic activities. In addition, as this structurally non-proteolytic feature is unique to the calpain-6 of placental mammals among vertebrates, our mutant mice might provide insight into the relationship between molecular and biological evolution.
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影响因子：
5.3
作者：
Arthur, JSC;Elce, JS;Greer, PA
通讯作者：
Greer, PA
影响因子：
2.9
作者：
BABAI, F;MUSEVIAGHDAM, J;GABBIANI, G
通讯作者：
GABBIANI, G
影响因子：
4.5
作者：
Mahajan VB;Skeie JM;Bassuk AG;Fingert JH;Braun TA;Daggett HT;Folk JC;Sheffield VC;Stone EM
通讯作者：
Stone EM
影响因子：
--
作者：
Dutt P;Croall DE;Arthur JS;Veyra TD;Williams K;Elce JS;Greer PA
通讯作者：
Greer PA
影响因子：
64.8
作者：
Irmler, M;Thome, M;Tschopp, J
通讯作者：
Tschopp, J