Endogenous expression and localization of myostatin and its relation to myosin heavy chain distribution in C2C12 skeletal muscle cells
Endogenous expression and localization of myostatin and its relation to myosin heavy chain distribution in C2C12 skeletal muscle cells
复制标题
DOI：
10.1002/jcp.10044
复制
发表时间：
2002-02-01
影响因子：
5.6
通讯作者：
Gonzalez-Cadavid, NF
中科院分区：
文献类型：
--
作者：
Artaza, JN;Bhasin, S;Gonzalez-Cadavid, NF
肌肉生长抑制素是骨骼肌生长的负调控因子。我们以前曾报道过重组的myostatin蛋白抑制C2C12细胞的DNA和蛋白质的合成。我们的目的是检测C2C12细胞是否表达Myostatin，确定其在C2C12细胞中的亚细胞定位，以及表达myostatin基因和蛋白的C2C2细胞的发育阶段。为研究肌抑素的内源性表达，将C2C12成肌细胞培养成肌管，用RT-PCR方法检测内源性肌抑素mRNA表达水平的变化。用免疫印迹法检测肌球蛋白重链(MHC)亚型(MHC-1和MHC-11)和肌肉生长抑素蛋白的表达。用改良的聚丙烯酰胺凝胶电泳法(PAGE)对MHC的相对表达模式进行确认。免疫荧光染色定位Myostatin的表达部位和MHC亚型的相对分布。用双重染色方法研究了这些蛋白的共表达。Myostatin rnRNA在肌管中有表达，在成肌细胞中未见表达。在表达MHC-11的多核纤维中，Myostatin蛋白存在于大部分但不是全部的细胞核中，并且在肌管的胞浆中也检测到了myostatin。分离的核组分和胞浆组分的Western印迹结果证实了Myostatin蛋白在肌管细胞核中的定位。C2C12肌管与地塞米松分级克隆孵育后，可剂量依赖地增加肌抑素的核免疫染色强度，并导致胞浆表达。综上所述，Myostatin主要在表达MHC-11的C2C12肌管细胞核中表达。其主要的核定位表明它可能在转录调控中发挥作用。J.细胞。物理。190：170-179,2002。(C)2002年Wiley-Liss，Inc.
Myostatin is a negative regulator of skeletal muscle growth. We have previously reported that recombinant myostatin protein inhibits DNA and protein synthesis in C2C12 cells. Our objective was to assess if C2C12 cells express myostatin, determine its sub-cellular localization and the developmental stage of C2C1 2 cells in which myostatin mRNA and protein are expressed. To study the endogenous expression of myostatin, C2C12 myoblasts were allowed to progress to myotubes, and changes in the levels of endogenous myostatin mRNA expression were determined by RT-PCR. The myostatin protein and the two major myosin heavy chain (MHC) isoforms (MHC-1 and -11) were determined by Western blot. Confirmation of the relative MHC expression patterns was obtained by a modified polyacrylamide gel electropheretic (PAGE) procedure. Immunofluorescence staining was employed to localize the site of myostatin expression and the relative distribution of the MHC isoforms. Co-expression of these proteins was studied using a dual staining approach. Expression of myostatin rnRNA was found in myotubes but not in myoblasts. Myostatin protein was seen in most but not all, of the nuclei of polynucleated fibers expressing MHC-11, and myoostatin was detected in the cytoplasm of myotube. The localization of myostatin protein in myotube nuclei was confirmed by Western blot of isolated nuclear and cytoplasmic fractions. Incubation of C2C1 2 myotubes with graded clones of dexamethasone dose-dependently increased the intensity of nuclear myostatin immunostaining and also resulted in the appearance of cytoplasmic expression. In conclusion, myostatin was expressed mostly in C2C12 myotubes nuclei expressing MHC-11. Its predominant nuclear localization suggests that it may play a role in transcriptional regulation. J. Cell. Physiol. 190: 170-179, 2002. (C) 2002 Wiley-Liss, Inc.