Mustn1 is expressed during chondrogenesis and is necessary for chondrocyte proliferation and differentiation in vitro.
Mustn1 is expressed during chondrogenesis and is necessary for chondrocyte proliferation and differentiation in vitro.
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DOI：
10.1016/j.bone.2009.04.245
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发表时间：
2009-08
期刊：
影响因子：
4.1
通讯作者：
Hadjiargyrou, Michael
中科院分区：
文献类型：
--
作者：
Gersch, Robert P.;Hadjiargyrou, Michael
Mustn1编码一种在肌肉骨骼系统中特异表达的小核蛋白，最初被认为是在骨再生过程中强烈上调的基因，特别是在骨折骨痂增殖的软骨细胞中。进一步的实验研究了它在软骨形成过程中的表达和作用。最初，进行了整装小鼠的原位杂交，发现Mustn1在活跃的软骨形成区域表达，包括四肢芽、鳃弓和尾芽。为了阐明Mustn1的功能，在软骨细胞前细胞系RCJ3.1C5.18(RCJ)中进行了过表达和沉默Mustn1的实验。在这些细胞中，Mustn1通常受到分化调控，在诱导分化2天后表达高峰。此外，Mustn1在多个RCJ细胞系中成功地过表达了~2-6倍，而在沉默的细胞系中与亲本的Mustn1水平相比降低了~32-52%。检测过表达、沉默、对照和亲本RCJ细胞系的增殖和分化。当Mustn1高表达系与亲本和对照相比时，在增殖和蛋白多糖产生方面没有观察到统计上的显著变化。相比之下，Mustn1沉默的细胞系的增殖率和分化程度均显著降低。具体地说，与亲本和随机对照相比，RNAi沉默的细胞系的种群数量减少了约55%-75%，基质(蛋白多糖)的产量也减少了约34%-40%。此外，基质产量的减少伴随着软骨形成标志基因的显著下调，如Sox9、II型胶原(Col II)和X型胶原(Col X)。最后，将Mustn1基因重新导入沉默的细胞系，挽救了这种表型，使增殖率、基质产生和软骨形成标记基因的表达恢复到亲代水平。综上所述，这些数据表明Mustn1是软骨细胞功能的必要调节因子。
Mustn1 encodes a small nuclear protein expressed specifically in the musculoskeletal system that was originally identified as a strongly up-regulated gene during bone regeneration, especially in fracture callus proliferating chondrocytes. Further experiments were undertaken to investigate its expression and role during chondrogenesis. Initially, whole mount mouse in situ hybridization was carried out and revealed Mustn1 expression in areas of active chondrogenesis that included limb buds, branchial arches and tail bud. To elucidate its function, experiments were carried out to perturb Mustn1 by overexpression and silencing in the pre-chondrocytic RCJ3.1C5.18 (RCJ) cell line. In these cells, Mustn1 is normally differentially regulated, with a spike in expression 2 days after induction of differentiation. Further, Mustn1 was successfully overexpressed in multiple RCJ cell lines by ~2–6 fold, and reduced to ~32–52% in silenced cell lines as compared to parental Mustn1 levels. Overexpressing, silenced, control, and parental RCJ cell lines were assayed for proliferation and differentiation. No statistically significant changes were observed in either proliferation or proteoglycan production when Mustn1 overexpressing lines were compared to parental and control. By contrast, both proliferation rate and differentiation were significantly reduced in Mustn1 silenced cell lines. Specifically, RNAi silenced cell lines showed reductions in populations of ~55–75%, and also ~34–40% less matrix (proteoglycan) production as compared to parental and random control lines. Further, this reduction in matrix production was accompanied by significant downregulation of chondrogenic marker genes, such as Sox9, Collagen type II (Col II), and Collagen type X (Col X). Lastly, reintroduction of Mustn1 into a silenced cell line rescued this phenotype, returning proliferation rate, matrix production, and chondrogenic marker gene expression back to parental levels. Taken together these data suggest that Mustn1 is a necessary regulator of chondrocyte function.
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