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刺牛(自发性肌肉抑制素缺陷型牛：DM牛)双肌肉表型导致肌肉质量增加的全部机制。1)在胸长肌成肌细胞培养中，肌肉生长抑制素抑制IGF-2的表达，但不抑制IGF-1的表达，而促成肌细胞增殖的HGF和aFGF促进其表达。但Myostatin不影响HGF的表达。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肝细胞生长因子产生的肌肉生长抑制素缺乏。2)通过基因芯片分析，与肌肉生长抑制素细胞内信号通路相关的转化生长因子-β诱导的早期基因家族2(TIEG-2)在DM牛中的表达是对照牛的4.5倍。…对TIEG-1基因的敲除更多的siRNA导致细胞生长和肌管形成增加，进而增强MyoD家族、MRF4和IGF-2的表达。这些结果表明TIEG-1与细胞内信号转导密切相关。3)首先从DM成肌细胞建立了9种克隆的DM成肌细胞系(DMC)，然后从DMC中获得了永生克隆的DM成肌细胞系(DMC-t)。这些细胞系非常便于准确了解肌肉抑制素在肌肉分化和生长中的作用。4)在IGF-1基因敲除小鼠和野生型小鼠的肌肉中也检测到类似的Myostatin。DM牛胸长肌中IGF-2的表达强于对照牛。Myostatin抑制DMC中IGF-2的表达，但对IGF-1的表达无影响。而活化素受体IIB型(ActRIIB)为myostatin受体和IGF-1受体的表达不受myostatin的影响，结果表明myostatin和IGF-2相互作用，myostatin和IGF-1独立作用于肌肉的分化和再生。5)免疫组织化学显示，在牛和猪腺垂体中，myostatin主要存在于促甲状腺细胞中，而ActRIIB仅存在于促肾上腺皮质激素细胞中。肌抑素阳性的甲状腺激素细胞多位于ActRIIB阳性的促肾上腺皮质激素细胞附近。6)小鼠ACTH腺瘤细胞株、AtT-20细胞表达ActRIIB。在AtT-20细胞培养的基础上加入肌肉生长抑素，细胞中POMC的表达和ACTH的免疫反应性被显著抑制。结果表明，MSTN直接抑制ACTH细胞中ACTH的产生。本研究按计划进行，获得了有助于全面了解肌抑制蛋白在肌肉分化和生长中作用的结果。较少

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