Reduction in vinculin levels with Rb1 loss is responsible for altered differentiation in preosteoblasts

IF 3.5 3区 生物学 Q3 CELL BIOLOGY
Elisha Pendleton, Keren Abdallah, Nalini Chandar
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引用次数: 0

Abstract

We have shown previously that loss of Rb1 in osteoblasts results in changes to gap junctional intercellular communication and a decrease in membrane proteins such as connexin 43 and cadherin 11. These cells also start expressing adipocyte specific transcription factors and marker genes while continuing to express osteoblast specific differentiated features. The mechanistic reason for this shift in differentiation behavior is not known. Recent studies have suggested a role for vinculin, a protein component of focal adhesions, in the transduction of signals to YAP-TAZ transcription factors to suppress adipocyte differentiation and allow osteoblast differentiation. We found loss of Rb1 expression in osteoblasts dramatically reduced vinculin quantity, with less distribution within focal adhesions when tested by immunofluorescence. Vinculin levels steadily increased during in vitro osteoblast differentiation and were about 6-8-fold higher in differentiated osteoblasts. Rb1 deficient cells showed an increase albeit at reduced levels (2-4-fold) during differentiation. A corresponding increase in YAP-TAZ activity and expression were seen in control osteoblasts and a stunted but similar response was present in Rb1 deficient osteoblasts. The fact that Rb1 deficient osteoblasts respond by increasing vinculin levels suggests that they can maintain the expected functional changes at a reduced level. To demonstrate that the concentration of vinculin was the determining factor, we either over expressed or knocked down vinculin which resulted in appropriately altering YAP/TAZ activity. Increased vinculin expression enhanced osteogenic while inhibiting adipocytic gene expression. During osteoblast differentiation, TAZ can be clearly visualized in the nucleus in osteoblasts’ whereas with Rb1 deficiency the distribution was diffuse within the cells. The reduction in vinculin expression, combined with a feeble response in YAP-TAZ signaling activity, may explain the lack of complete inhibition of adipocyte specific gene expression and a generation of a mixed phenotype seen with loss of Rb1 function. As the adipocyte master transcription factor PPAR-gamma levels are increased by Rb1 loss, we knockdown PPAR-gamma which resulted in an increase to vinculin expression, showing that the PPAR-gamma induced activation of adipocyte differentiation pathway is responsible for reducing vinculin expression in RbKD cells.
血管蛋白水平降低与Rb1缺失是成骨前细胞分化改变的原因
我们之前已经证明,成骨细胞中Rb1的缺失会导致间隙连接细胞间通讯的改变和膜蛋白(如连接蛋白43和钙粘蛋白11)的减少。这些细胞也开始表达脂肪细胞特异性转录因子和标记基因,同时继续表达成骨细胞特异性分化特征。这种分化行为转变的机制原因尚不清楚。最近的研究表明,局灶黏附的蛋白质成分vinculin在向YAP-TAZ转录因子传递信号以抑制脂肪细胞分化并允许成骨细胞分化中的作用。我们发现,在成骨细胞中Rb1表达的缺失显著降低了血管蛋白的数量,免疫荧光检测时发现,在局灶粘连中分布较少。在体外成骨细胞分化过程中,血管素水平稳步上升,在分化成骨细胞中,血管素水平高出约6-8倍。Rb1缺陷细胞在分化过程中表现出增加,尽管水平降低(2-4倍)。在对照成骨细胞中,YAP-TAZ的活性和表达相应增加,而在Rb1缺乏的成骨细胞中,YAP-TAZ的活性和表达也相应增加。Rb1缺乏的成骨细胞通过增加血管蛋白水平作出反应,这表明它们可以在较低的水平上维持预期的功能变化。为了证明vinculin的浓度是决定因素,我们要么过表达或敲低vinculin,导致适当改变YAP/TAZ活性。血管蛋白表达增加促进成骨,同时抑制脂肪细胞基因表达。在成骨细胞分化过程中,TAZ在成骨细胞细胞核内清晰可见,而Rb1缺乏时,TAZ在细胞内呈弥漫性分布。vinculin表达的减少,加上YAP-TAZ信号活性的微弱反应,可能解释了脂肪细胞特异性基因表达缺乏完全抑制和Rb1功能丧失所见的混合表型的产生。由于脂肪细胞主转录因子PPAR-gamma水平因Rb1缺失而升高,我们敲低PPAR-gamma导致血管素表达增加,表明PPAR-gamma诱导的脂肪细胞分化途径的激活是降低RbKD细胞中血管素表达的原因。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Experimental cell research
Experimental cell research 医学-细胞生物学
CiteScore
7.20
自引率
0.00%
发文量
295
审稿时长
30 days
期刊介绍: Our scope includes but is not limited to areas such as: Chromosome biology; Chromatin and epigenetics; DNA repair; Gene regulation; Nuclear import-export; RNA processing; Non-coding RNAs; Organelle biology; The cytoskeleton; Intracellular trafficking; Cell-cell and cell-matrix interactions; Cell motility and migration; Cell proliferation; Cellular differentiation; Signal transduction; Programmed cell death.
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