Effects of myostatin, a negative regulator of muscle mass, that modulates and improves beef production

本研究旨在分析日本短角牛（自发性肌生长抑制素缺乏牛：DM牛）双肌表型诱导肌肉质量增加的整个机制。本研究从2005年至2007年进行了三年的研究，取得了以下新的研究成果：1）在M.在DM牛胸最长肌中，myostatin抑制IGF-2的表达，但不抑制IGF-1的表达，而刺激成肌细胞增殖的HGF和aFGF则促进myostatin的表达。而肌生成抑制素对肝细胞生长因子的表达无影响。这些结果表明，DM牛肌肉质量的增加在很大程度上依赖于成肌细胞产生的HGF产生的肌生成抑制素的缺陷。2）通过微阵列分析，确定了与肌生成抑制素细胞内信号通路相关的TGF-β诱导型早期基因家族2（TIEG-2）在DM牛中的表达是对照牛的4.5倍。TIEG-1的敲除， ...更多信息 siRNA诱导细胞生长和肌管形成增加，并进一步增强MyoD家族、MRF 4和IGF-2的表达。这些结果表明，TIEG-1作为一种特异性因子与肌生成抑制素细胞内信号转导密切相关。3）从DM成肌细胞出发，建立了9种DM成肌细胞克隆株（DMc），并从DMc出发，建立了永生化DM成肌细胞克隆株（DMc-t）。这些细胞系非常便于精确地理解肌肉生长抑制素对肌肉分化和生长的作用。4）在IGF-1敲除小鼠和野生型小鼠的肌肉中类似地检测到肌肉生长抑制素。IGF-2在M. DM牛的胸最长肌比对照牛的胸最长肌长。在DMc培养物中，肌肉生长抑制素抑制IGF-2的表达，而对IGF-1的表达没有影响。而激活素Ⅱ B型受体（ActRIIB）作为肌肉生长抑制素受体和IGF-1受体的表达不受肌肉生长抑制素的影响。结果表明，肌肉生长抑制素和IGF-2共同作用，肌肉生长抑制素和IGF-1独立作用于肌肉的分化和再生。免疫组化结果显示，牛腺垂体中肌生长抑制素主要存在于促甲状腺细胞中，ActRIIB主要存在于促肾上腺皮质细胞中，猪大量肌生成抑制素阳性的促甲状腺细胞位于ActRIIB阳性的促肾上腺皮质细胞的附近。结果表明，促甲状腺激素细胞释放的肌肉生长抑制素通过旁分泌途径作用于促肾上腺皮质激素细胞。6）小鼠ACTH腺瘤细胞株AtT-20细胞表达ActRIIB。肌生长抑制素除了AtT-20细胞培养物中，POMC的表达和ACTH免疫反应性的细胞基本上被抑制。本研究首次发现肌生长抑制剂直接抑制ACTH细胞的ACTH生成，为深入了解肌生长抑制剂对肌肉分化和生长的作用提供了实验依据。少

The present study was conducted to analyze the entire mechanisms that double muscled phenotype of Japanese shorthorn cattle (spontaneous myostatin-deficient cattle: DM cattle) induces the increased muscle mass. The research for three years form 2005 to 2007 resulted in several new information as follows.1) In the myoblast (DM myoblast) cultures derived from the M. longissimus thoracis of DM cattle, myostatin suppressed the expression of IGF-2 but not IGF-1, and HGF and aFGF that stimulate myoblast proliferation augmented the myostatin expression. But myostatin did not influence the HGF expression. These findings indicate that the increased muscle mass in DM cattle largely depends on deficient myostatin production by HGF produced in myoblasts.2) By microarray analysis, it was defined that the expression of TGF-β inducible early gene family 2 (TIEG-2) that is associated with myostatin intracellular signal pathway was 4.5 times more in DM cattle than control cattle. Knockdown of TIEG-1 by … More siRNA led the increased cell growth and myotube formation, and further the enhanced expression of number of MyoD family, MRF4 and IGF-2. These findings indicate that TIEG-1 is closely associated with myostatin intracellular signaling as one of specific factors.3) Nine kinds of cloned DM myoblast cell line (DMc) were first established from DM myoblasts and then the inmortal cloned DM myoblast cell line (DMc-t) was produced from DMc. These cell lines are very convenient to precisely understand myostatin action on muscle differentiation and growth.4) Myostatin was similarly detected in the muscle of IGF-1 knockout and wild type mice. IGF-2 expression was stronger in the M. longissimus thoracis of DM cattle than control cattle than control cattle. Myostatin suppressed IGF-2 expession and was no effect on IGF-1 expression in DMc cultures. But the expression of activin receptor type IIB (ActRIIB) to be myostatin receptor and IGF-1 receptor was not influenced by myostatin The results show that myostatin and IGF-2 reciprocally act and myostatin and IGF-1 independently act on muscle differentiation and regeneration of muscle.5) Immunohistochemistry revealed that myostatin was mainly contained in thyrotrophs and ActRIIB was detected in corticotrophs in the adenohypophysis of cattle and pigs. A lot of thyrotrophs positive for myostatin was located adjacently the corticotrophs positive for ActRIIB. The findings show that myostatin released from thyrotrophs acts on corticotrophs by paracrine pathway.6) Mouse ACTH adenoma cell line, AtT-20 cells were expressed ActRIIB. By myostatin addition to the AtT-20 cell cultures, the POMC expression and ACTH immunoreactivity in the cells were substantially suppressed. The results first show that MSTN directly suppresses the ACTH production in ACTH cells.The present study was proceeded according to schedules and obtained the results available to fully understand myostain action on muscle differentiation and growth. Less