Analysis of human skeletal muscle after 48 h immobilization reveals alterations in mRNA and protein for extracellular matrix components
Analysis of human skeletal muscle after 48 h immobilization reveals alterations in mRNA and protein for extracellular matrix components
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DOI：
10.1152/japplphysiol.00180.2006
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发表时间：
2006-10-01
影响因子：
3.3
通讯作者：
Clarkson, Priscilla M.
中科院分区：
文献类型：
--
作者：
L. Urso, Maria;Scrimgeour, Angus G.;Clarkson, Priscilla M.
我们研究了48小时的膝关节制动对人骨骼肌中mRNA和蛋白质变化的影响。我们假设，48小时的固定化将增加基因表达和泛素-蛋白酶体途径（UPP）组件各自的蛋白质产物。此外，我们使用微阵列分析来确定新的途径。在固定48小时前后，从5名男性（20.4 +/- 0.5岁）的股肌中取出活检组织。基因表达的整体变化通过使用Affyssin GeneChips进行分析。通过定量RT-PCR确认候选基因。蛋白质印迹法（WB）用于定量候选基因的蛋白质产物并评估Akt途径活化。免疫组织化学用于定位蛋白质，发现改变时，通过WB评估。用基因芯片显示改变表达的最大百分比的基因包括涉及UPP、金属硫蛋白功能和细胞外基质（ECM）完整性的基因。定量RT-PCR分析证实了UPP组分[ USP-6，小泛素相关修饰剂（SUMO-1）]和金属硫蛋白（MT 2A，MT 1F，MT 1H，MT 1X）的mRNA增加，基质金属蛋白酶（MMP-28，TIMP-1）和ECM结构组分[胶原蛋白III（COLIII）和IV（COLIV）]的mRNA含量降低。只有磷酸化Akt（Ser 473，Thr 308），COLIII和COLIV蛋白水平有显着不同的制动后（分别下降25，10，88，和28%）。免疫组织化学证实WB显示胶原蛋白固定后染色减少。我们的研究结果表明，48小时的固定增加mRNA含量的UPP和金属硫蛋白功能的组件，而减少mRNA和蛋白质的ECM组件，以及减少Akt的磷酸化。
We examined the effects of 48 h of knee immobilization on alterations in mRNA and protein in human skeletal muscle. We hypothesized that 48 h of immobilization would increase gene expression and respective protein products for ubiquitin-proteasome pathway (UPP) components. Also, we used microarray analysis to identify novel pathways. Biopsies were taken from the vastus muscle of five men (20.4 +/- 0.5 yr) before and after 48-h immobilization. Global changes in gene expression were analyzed by use of Affymetrix GeneChips. Candidate genes were confirmed via quantitative RT-PCR. Western blotting (WB) was used to quantify protein products of candidate genes and to assess Akt pathway activation. Immunohistochemistry was used to localize proteins found to be altered when assessed via WB. The greatest percentage of genes showing altered expression with the GeneChip included genes involved in the UPP, metallothionein function, and extracellular matrix (ECM) integrity. Quantitative RT-PCR analysis confirmed increases in mRNA for UPP components [ USP-6, small ubiquitin-related modifier (SUMO-1)] and the metallothioneins (MT2A, MT1F, MT1H, MT1X) and decreases in mRNA content for matrix metalloproteinases (MMP-28, TIMP-1) and ECM structural components [ collagen III (COLIII) and IV (COLIV)]. Only phosphorylated Akt (Ser473, Thr308), COLIII and COLIV protein levels were significantly different postimmobilization (25, 10, 88, and 28% decrease, respectively). Immunohistochemistry confirmed WB showing decreased staining for collagens postimmobilization. Our results suggest that 48 h of immobilization increases mRNA content for components of the UPP and metallothionein function while decreasing mRNA and protein for ECM components as well as decreased phosphorylation of Akt.